TWI818916B - Anti cd147 antibody, use and manufacturing method thereof - Google Patents

Abstract

The subject of the present invention is to provide a novel anti-CD147 antibody that exhibits excellent anti-tumor activity and is excellent in safety. Another subject of the present invention is to provide pharmaceuticals containing such antibodies. Another object of the present invention is to provide a method of treating tumors using the antibody or pharmaceutical.

According to the present invention, CD147-specific antibodies are provided that activate CD147 and exhibit high anti-tumor effects. According to the present invention, an anti-CD147 antibody that exhibits high anti-tumor effect independent of effector function is provided. According to the present invention, a pharmaceutical composition containing such an anti-CD147 antibody is provided. According to the present invention, a method for treating tumors using such anti-CD147 antibodies and/or pharmaceutical compositions is provided.

Description

Anti-CD147 antibodies, uses and manufacturing methods thereof

The present invention relates to an anti-CD147 antibody showing excellent anti-tumor effect, a method for producing the anti-CD147 antibody, an anti-tumor agent containing the anti-CD147 antibody, and the like.

It is known that due to advances in cancer treatment methods and therapeutic drugs, cancer that was once considered an incurable disease can be treated and cured. CTLA4 antibodies and PD1 antibodies, developed as antibody drugs showing particularly excellent drug stability and specificity, produce high response rates against melanoma and some solid cancers through the activation of immune cells including T cells. , or there may be a cure, which becomes good news for cancer patients. Although these therapeutic drugs have been tried for the treatment of many refractory solid cancers, most cancers such as pancreatic cancer and liver cancer do not show sensitivity to drugs, and even if they are removed by surgery, Or in the past, anti-cancer drugs were the basic treatment, but high-frequency relapses have also been observed, and there is a clear need for treatments and drugs that can bring about a radical cure.

CD147 is a primary transmembrane protein with 2 to 3 immunoglobulin-like domains. It is known that CD147 interacts with each other and interacts with CD44 and integrin (Integrin) family molecules. , CD98, VEGFR, CypA/B, MCT1/3/4 and other extracellular and cell membrane surface molecules involved in proliferation, infiltration, inflammation, etc., resulting in downstream message-related molecules, FAK, MEK, Erk, JAK/STAT, AKT , activate MAPK family molecules, promote the production of proteases headed by MMP, and promote cancer proliferation, metastasis, and invasion. In addition, for the above-mentioned liver cancer and pancreatic cancer patients, it has been reported that if CD147 expression is high, the survival period is short and the prognosis is poor, and it is considered to be one of the target molecules for cancer treatment.

As for antibodies targeting CD147, ABX-CBL and licartin have been actually administered to humans clinically. ABX-CBL not only inhibits the binding of CD147 to cyclophilin A (cyclophilin A) and inhibits the activity of T cells, but also shows complement-dependent cytocidal activity in the blood against normal cells including CD147-positive T cells. Although clinical trials were conducted targeting GVHD, the drug efficacy was insufficient and severe muscle pain was observed, and it has not yet been approved as a drug (Patent Document 1, Non-Patent Document 1).

Licatin is a biological agent in which the radioactive isotope iodine I 131 is added to the Fab'2 portion of the HAb18 antibody, and has been approved as a cancer type suitable for liver cancer in China (Non-patent Document 2, Non-Patent Document 3). Licatin lacks the Fc part of the antibody that activates immune cells or complement. Although there are no reports of immune-mediated toxicity, there are no reports of clinical cure of liver cancer.

In addition, as for other antibodies targeting CD147, anti-CD147 monoclonal antibodies are known to block biological activities related to CD147 such as angiogenesis and VEGF production, matrix metalloproteinase, etc. (Patent Document 2); Anti-CD147 monoclonal antibodies that inhibit the activation of T cells (Non-Patent Document 4); and antibodies that specifically bind to CD147 molecules characterized by having ADCC activity and CDC activity (Patent Document 3). However, there is no known CD147 antibody that does not have effector function and exhibits anti-tumor effect. Furthermore, the relationship between the activation of the cell signaling system through CD147 and the anti-tumor effect is unknown.

Prior technical literature patent documents

Patent Document 1 WO1999/045031

Patent document 2 WO2010/036460

Patent document 3 WO2017/061602

non-patent literature

Non-patent literature 1 Deeg H, et al., J. Blood., 2052-2058, 2001

Non-patent literature 2 Chen Z, et al., Int. J. Radiation Oncology Biol. Phys., 435-444, 2006

Non-patent literature 3 Xu J, et al., Hepatology, 269-276, 2007

Non-patent literature 4 Koch C, et al., International Immunology, 777-786, 1999

Summary of the invention

The subject of the present invention is to provide novel anti-CD147 antibodies with novel pharmacological effects, excellent safety, and high anti-tumor effect; pharmaceuticals containing the antibodies; and methods of treating tumors using the antibodies or pharmaceuticals.

The inventors of the present invention conducted in-depth examinations to achieve the above-mentioned problems, and discovered for the first time that the activation of signaling molecules related to CD147 is related to the anti-tumor effect. Furthermore, the present inventors succeeded in obtaining a CD147-specific antibody that activates CD147 and exhibits high anti-tumor effect. The antibody of the present invention has the characteristic of exhibiting high anti-tumor effect independent of effector function. In addition, although there have been reports so far of antibodies showing anti-tumor effects depending on effector functions, the antibody system of the present invention has the characteristics of not acting on T cells and PBMCs, and also showing high anti-tumor effects independent of effector functions. It is an antibody that is expected to have excellent safety as a pharmaceutical. The antibody of the present invention is bound to liver cancer cells and exhibits significantly stronger efficacy than sorafenib, one of the standard therapeutic drugs for liver cancer. The antibody of the present invention is bound to pancreatic cancer cells and exhibits significantly stronger efficacy than gemcitabine, one of the standard therapeutic drugs for pancreatic cancer. Furthermore, the antibody of the present invention binds to chronic myelogenous leukemia cells and exhibits significantly stronger efficacy than imatinib, one of the standard therapeutic drugs for chronic myelogenous leukemia. The present inventors have discovered that the CD147 antibody system of the present invention activates the p38MAPK and SMAD signaling systems in cancer cells. Furthermore, the present inventors have discovered that the CD147 antibody system of the present invention exhibits excellent anti-tumor effects on SMAD4-positive cells.

The present invention includes the following inventions.

[1] A human CD147 antibody or an antigen-binding fragment of the antibody, characterized by competing for binding to human CD147 with at least one antibody selected from the group consisting of (A) to (F) below, and Through the signaling activation of CD147, (A) includes a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 71, and a light chain variable region composed of the amino acid sequence represented by SEQ ID NO: 69. An antibody with a chain variable region, (B) a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 51, and a light chain composed of the amino acid sequence represented by SEQ ID NO: 49 The variable region antibody (C) includes a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 61, and a light chain composed of the amino acid sequence represented by SEQ ID NO: 59. Antibodies with variable regions, (D) include a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 81, and a light chain variable region composed of the amino acid sequence represented by SEQ ID NO: 79 The antibody of the region, (E) contains a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 10, and a light chain variable region composed of the amino acid sequence represented by SEQ ID NO: 8 The antibody, and (F) includes a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 20, and a light chain variable region composed of the amino acid sequence represented by SEQ ID NO: 18 of antibodies.

[2] A human CD147 antibody or an antigen-binding fragment of the antibody, characterized by binding to an epitope bound by at least one antibody selected from the group consisting of (A) to (F) below , and activates signaling through CD147, (A) includes a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 71, and a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 69 An antibody consisting of a light chain variable region, (B) a heavy chain variable region composed of an amino acid sequence represented by SEQ ID NO: 51, and a heavy chain variable region composed of an amino acid sequence represented by SEQ ID NO: 49 An antibody with a light chain variable region, (C) a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 61, and an antibody consisting of an amino acid sequence represented by SEQ ID NO: 59 An antibody with a light chain variable region, (D) including a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 81, and a light chain variable region composed of the amino acid sequence represented by SEQ ID NO: 79 An antibody with a chain variable region, (E) including a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 10, and a light chain composed of the amino acid sequence represented by SEQ ID NO: 8 Antibodies with variable regions, and (F) a heavy chain variable region composed of the amino acid sequence represented by SEQ ID NO: 20, and a light chain composed of the amino acid sequence represented by SEQ ID NO: 18 Antibodies with variable regions.

[3] The human CD147 antibody or the antigen-binding fragment of the antibody as described in [1] or [2], the ADCC activity is reduced or missing.

[4] The CDC activity of the human CD147 antibody or the antigen-binding fragment of the antibody described in any one of [1] to [3] is reduced or missing.

[5] The ADCP activity of the human CD147 antibody or the antigen-binding fragment of the antibody described in any one of [1] to [4] is reduced or missing.

[6] The antibody or the antigen-binding fragment of the antibody as described in any one of [1] to [5], which contains arginine (Arg) from No. 106 to No. 165 of SEQ ID NO. 3 Binds to the epitope of glycine (Gly) residues.

[7] The antibody or antigen-binding fragment of the antibody as described in any one of [1] to [6], which binds to the following epitope: No. 106 of the amino acid sequence described in SEQ ID NO: 3 No. arginine (Arg), No. 108 lysine (Lys), No. 109 alanine (Ala), No. 110 valine (Val), No. 127 lysine (Lys) ), No. 128 Serine (Ser), No. 129 Glutamic acid (Glu), No. 130 Serine (Ser), No. 131 Valine (Val), No. 132 Proline (Pro), No. 133 Proline (Pro), No. 134 Valine (Val), No. 164 Glutamine (Gln) and No. 165 Glycine (Gly ) of each residue.

[8] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [7], characterized in that the heavy chain sequence includes a variable region having CDRH1, CDRH2 and CDRH3, and the aforementioned CDRH1 is composed of the sequence The aforementioned CDRH2 is composed of the amino acid sequence represented by the sequence identification number 75, and the aforementioned CDRH3 is composed of the amino acid sequence represented by the sequence identification number 77; and The light chain sequence includes variable regions having CDRL1, CDRL2, and CDRL3. The aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 72, and the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 73. , the aforementioned CDRL3 is composed of the amino acid sequence shown in Sequence ID No. 74.

[9] The antibody as described in any one of [1] to [5] or the antigen-binding fragment of the antibody, which contains the amino acid sequence described in SEQ ID NO: 143, or is in the sequence of SEQ ID NO: 143 Antitope binding of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added.

[10] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [5] or 9, characterized in that the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, and the aforementioned CDRH1 is composed of the amino acid sequence represented by SEQ ID NO: 55, the aforementioned CDRH2 is composed of the amino acid sequence represented by SEQ ID NO: 56, and the aforementioned CDRH3 is composed of the amino acid sequence represented by SEQ ID NO: 57 ; and the light chain sequence includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 52, and the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 53 The aforementioned CDRL3 is composed of the amino acid sequence shown in Sequence ID No. 54.

[11] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [5] or 9, characterized in that the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, and the aforementioned CDRH1 is composed of the amino acid sequence represented by SEQ ID NO: 65, the aforementioned CDRH2 is composed of the amino acid sequence represented by SEQ ID NO: 66, and the aforementioned CDRH3 is composed of the amino acid sequence represented by SEQ ID NO: 67 Composition; and the light chain sequence includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 62, and the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 63 The aforementioned CDRL3 is composed of the amino acid sequence shown in Sequence ID No. 64.

[12] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [5] or 9, characterized in that the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, and the aforementioned CDRH1 is It consists of the amino acid sequence represented by SEQ ID NO. 85, the aforementioned CDRH2 consists of the amino acid sequence represented by SEQ ID NO. 86, and the aforementioned CDRH3 consists of the amino acid sequence represented by SEQ ID NO. 87. ; and the light chain sequence includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 82, and the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 83 The aforementioned CDRL3 is composed of the amino acid sequence represented by Sequence ID No. 84.

[13] The antigen-binding fragment of the antibody according to any one of [1] to [12], which is selected from the group consisting of Fab, F(ab')2, Fab' and Fv.

[14] The antibody according to any one of [1] to [12], which is an scFv.

[15] The antibody according to any one of [1] to [12] or the antigen-binding fragment of the antibody, which is a chimeric antibody.

[16] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [12], which is characterized by being humanized.

[17] The antibody according to any one of [1] to [16], wherein the heavy chain is a constant region including a human immunoglobulin G1 heavy chain, a human immunoglobulin G2 heavy chain, or a human immunoglobulin G4 heavy chain. , and the light chain includes the constant region of the human immunoglobulin kappa light chain.

[18] The antibody according to [17], the heavy chain of which contains the constant region of the human immunoglobulin G4 heavy chain.

[19] The antibody according to [18], wherein in the constant region of the human immunoglobulin G4 heavy chain, serine (Ser) No. 228 shown in the EU index (EU index) is passed through proline (Pro )replace.

[20] The antibody of [18], in which the phenylalanine (Phe) of No. 234 shown in the EU index is replaced by alanine (Ala) in the constant region of the human immunoglobulin G4 heavy chain, and No. 235 No. leucine (Leu) was replaced by alanine (Ala).

[21] The antibody of [18], in which serine (Ser) at No. 228 shown in the EU index is replaced by proline (Pro) in the constant region of the human immunoglobulin G4 heavy chain. Phenylalanine (Phe) in No. 234 was replaced with alanine (Ala), and leucine (Leu) in No. 235 was replaced with alanine (Ala).

[22] The antibody according to [17], wherein the heavy chain contains the constant region of a human immunoglobulin G2 heavy chain.

[23] A human CD147 antibody or an antigen-binding fragment of the antibody, characterized by: having the following (c) and (d), and activating information transmission through CD147, (c) is selected from the group consisting of the following (c1 ) to (c4): (c1) Consisting of amino acid residues Nos. 20 to 136 of the amino acid sequence represented by SEQ ID NO: 135 Heavy chain variable region; (c2) A heavy chain variable region composed of amino acid residues No. 20 to 136 of the amino acid sequence shown in Sequence ID No. 147; (c3) in (c1) or The sequence of (c2) is an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (c4) (any one of (c1) ~ (c3) The sequence of the framework region other than each CDR sequence in the sequence described in the item, the amino acid sequence in which one or several amino acids are deleted, substituted or added, and (d) is selected from the group consisting of the following (d1)~ The light chain variable region described in any one of the groups (d5): (d1) A light chain composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 137 Variable region; (d2) A light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 149; (d3) represented by SEQ ID NO: 151 The light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence; (d4) The sequence described in any one of (d1) to (d3), except for each CDR sequence The sequence of the framework region has an amino acid sequence with at least 95% sequence identity; and (d5) the sequence of the framework region other than each CDR sequence in the sequence described in any one of (d1) to (d4) , an amino acid sequence in which one or several amino acids are deleted, substituted or added.

[24] The antibody or antigen-binding fragment of the antibody according to [23], which contains a heavy chain composed of amino acid residues No. 20 to 136 of the amino acid sequence represented by SEQ ID NO: 135 A variable region, and a light chain variable region composed of amino acid residues No. 21 to No. 128 of the amino acid sequence shown in Sequence ID No. 137.

[25] The antibody or antigen-binding fragment of the antibody as described in [23], which contains a heavy chain composed of amino acid residues No. 20 to 463 of the amino acid sequence represented by SEQ ID NO: 135 , and a light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in Sequence ID No. 137.

[26] The antibody or antigen-binding fragment of the antibody according to [23], which contains a heavy chain composed of amino acid residues No. 20 to 136 of the amino acid sequence represented by SEQ ID NO: 147 A variable region, and a light chain variable region composed of amino acid residues No. 21 to No. 128 of the amino acid sequence shown in SEQ ID NO: 149.

[27] The antibody or antigen-binding fragment of the antibody as described in [23], which contains a heavy chain composed of amino acid residues No. 20 to 463 of the amino acid sequence represented by SEQ ID NO: 147 , and a light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in Sequence ID No. 149.

[28] The antibody or antigen-binding fragment of the antibody according to [23], which contains a heavy chain composed of amino acid residues No. 20 to 136 of the amino acid sequence represented by SEQ ID NO: 147 A variable region, and a light chain variable region composed of amino acid residues No. 21 to No. 128 of the amino acid sequence shown in Sequence ID No. 151.

[29] The antibody or antigen-binding fragment of the antibody as described in [23], which contains a heavy chain composed of amino acid residues No. 20 to 463 of the amino acid sequence represented by SEQ ID NO: 147 , and a light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in Sequence ID No. 151.

[30] A human CD147 antibody or an antigen-binding fragment of the antibody, characterized by: having the following (a) and (b), and activating information transmission through CD147, (a) is selected from the group consisting of the following (a1 )~(a4) The heavy chain variable region described in any one of the groups: (a1) consisting of amino acid residues No. 20 to No. 140 of the amino acid sequence represented by Sequence ID No. 123 Heavy chain variable region; (a2) A heavy chain variable region composed of amino acid residues No. 20 to 140 of the amino acid sequence shown in SEQ ID NO: 125; (a3) in (a1) or The sequence of (a2) is an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (a4) the sequence described in any one of (a1) to (a3) The sequence of the framework region other than each CDR sequence in the amino acid sequence has one or more amino acids deleted, substituted or added, and (b) is selected from the group consisting of the following (b1) ~ (b3) The light chain variable region described in any one of the groups: (b1) a light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by Sequence ID No. 127; (b2) In the sequence of (b1), the amino acid sequence has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (b3) In the sequence of (b1) or (b2) In the sequence of the framework region other than each CDR sequence, one or several amino acids are deleted, substituted or added to the amino acid sequence.

[31] The antibody or antigen-binding fragment of the antibody as described in [30], which contains: a complex consisting of amino acid residues No. 20 to No. 140 of the amino acid sequence represented by Sequence ID No. 123. The chain variable region or the heavy chain variable region composed of amino acid residues No. 20 to 140 of the amino acid sequence represented by SEQ ID NO: 125, and the amino acid represented by SEQ ID NO: 127 The light chain variable region composed of amino acid residues No. 21 to 128 of the sequence.

[32] The antibody or the antigen-binding fragment of the antibody as described in [30], which contains: a complex consisting of amino acid residues No. 20 to No. 466 of the amino acid sequence represented by Sequence ID No. 123. chain or a heavy chain composed of amino acid residues Nos. 20 to 467 of the amino acid sequence represented by SEQ ID NO: 125, and a heavy chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence represented by SEQ ID NO: 127 A light chain composed of amino acid residues.

[33] A human CD147 antibody or an antigen-binding fragment of the antibody, characterized by: having the following (e) and (f), and activating information transmission through CD147, (e) is selected from the group consisting of the following (e1 )~(e4) The heavy chain variable region described in any one of the groups: (e1) consisting of amino acid residues Nos. 20 to 137 of the amino acid sequence represented by SEQ ID NO: 129 Heavy chain variable region; (e2) A heavy chain variable region composed of amino acid residues No. 20 to 137 of the amino acid sequence shown in Sequence ID No. 131; (e3) in (e1) or The sequence of (e2) is an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (e4) the sequence described in any one of (e1) to (e3) The sequence of the framework region other than each CDR sequence in the amino acid sequence has one or more amino acids deleted, substituted or added, and (f) is selected from the group consisting of the following (f1) ~ (f3) The light chain variable region described in any one of the groups: (f1) a light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by Sequence ID No. 133; (f2) in the sequence of (f1), an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (f3) in the sequence of (f1) or (f2) In the sequence of the framework region other than each CDR sequence, one or several amino acids are deleted, substituted or added to the amino acid sequence.

[34] The antibody or antigen-binding fragment of the antibody as described in [33], which contains: a complex consisting of amino acid residues No. 20 to No. 137 of the amino acid sequence represented by Sequence ID No. 129. The chain variable region or the heavy chain variable region composed of amino acid residues No. 20 to 137 of the amino acid sequence represented by SEQ ID NO: 131, and the amino acid represented by SEQ ID NO: 133 The light chain variable region composed of amino acid residues No. 21 to 128 of the sequence.

[35] The antibody or antigen-binding fragment of the antibody as described in [33], which contains: a complex consisting of amino acid residues No. 20 to No. 463 of the amino acid sequence represented by Sequence ID No. 129. chain or a heavy chain composed of amino acid residues Nos. 20 to 464 of the amino acid sequence represented by SEQ ID NO: 131, and a heavy chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence represented by SEQ ID NO: 133 A light chain composed of amino acid residues.

[36] A human CD147 antibody or an antigen-binding fragment of the antibody, characterized by: having the following (g) and (h), and activating information transmission through CD147, (g) is selected from the group consisting of the following (g1 )~(g3) The heavy chain variable region described in any one of the groups: (g1) consisting of amino acid residues Nos. 20 to 138 of the amino acid sequence represented by SEQ ID NO: 139 The heavy chain variable region; (g2) the sequence in (g1), the amino acid sequence having at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (g3) the sequence in (g1) or (g2) The sequence of the framework region other than each CDR sequence in the sequence, an amino acid sequence in which one or more amino acids are deleted, substituted or added, and (h) is selected from the group consisting of (h1) The light chain variable region described in any one of the groups ~(h3): (h1) A light chain consisting of amino acid residues Nos. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 141 chain variable region; (h2) the sequence in (h1), an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (h3) the sequence in (h1) or ( In the sequence of h2), the sequence of the framework region other than each CDR sequence has an amino acid sequence in which one or several amino acids have been deleted, substituted or added.

[37] The antibody or the antigen-binding fragment of the antibody as described in [36], which contains: a complex consisting of amino acid residues No. 20 to No. 138 of the amino acid sequence represented by Sequence ID No. 139. chain variable region, and a light chain variable region composed of amino acid residues Nos. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 141.

[38] The antibody or the antigen-binding fragment of the antibody as described in [36], which contains: a complex consisting of amino acid residues No. 20 to No. 464 of the amino acid sequence represented by Sequence ID No. 139. chain, and a light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in Sequence ID No. 141.

[39] The ADCC activity of the human CD147 antibody or the antigen-binding fragment of the antibody described in any one of [23] to [38] is reduced or missing.

[40] The CDC activity of the human CD147 antibody or the antigen-binding fragment of the antibody described in any one of [23] to [39] is reduced or missing.

[41] The human CD147 antibody or the antigen-binding fragment of the antibody described in any one of [23] to [40] has reduced or deleted ADCP activity.

[42] A pharmaceutical composition characterized by containing at least any one of the antibody described in any one of [1] to [41] or the antigen-binding fragment of the antibody.

[43] The pharmaceutical composition according to [42], which is an anti-tumor agent.

[44] The pharmaceutical composition according to [43], wherein the tumor is a tumor expressing CD147.

[45] The pharmaceutical composition as described in [43] or [44], wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, lymphoma, thyroid cancer, Skin cancer, head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumors, gastrointestinal stromal tumors (GIST), leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia ( CLL), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin lymphoma, or diffuse large cell B-cell lymphoma (DLBCL).

[46] The pharmaceutical composition according to any one of [43] to [45], wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, renal cancer, leukemia, acute myeloid leukemia (AML), chronic bone marrow chronic lymphocytic leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell B-cell lymphoma (DLBCL) ).

[47] The pharmaceutical composition according to any one of [43] to [46], wherein the tumor is a SMAD4-positive tumor or a tumor with reduced or deleted expression of KLF5.

[48] The pharmaceutical composition according to any one of [42] to [47], further containing other anti-tumor agents.

[49] A method of treating tumors, characterized by using the antibody or antigen-binding fragment of the antibody as described in any one of [1] to [41] or the antibody as described in any one of [42] to [48] The pharmaceutical composition is administered to the patient.

[50] The treatment method as described in [49], wherein the tumor is a tumor expressing CD147.

[51] The treatment method according to any one of [49] or [50], wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, lymphoma, Thyroid cancer, skin cancer, head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumors, gastrointestinal stromal tumors (GIST), leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell B-cell lymphoma (DLBCL).

[52] The treatment method as described in [49]~[51], wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, renal cancer, leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML) , chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell B-cell lymphoma (DLBCL).

[53] The treatment method according to any one of [49] to [52], wherein the tumor is a SMAD4-positive tumor or a tumor with reduced or deleted expression of KLF5.

[54] The treatment method according to any one of [49] to [53], which is administered in combination with another anti-tumor agent.

[55] A polynucleotide encoding the antibody described in any one of [1] to [41] or a functional fragment of the antibody.

[56] The polynucleotide described in [55], which includes a polynucleotide selected from the group consisting of (j1) to (j3) described in any one of the following: (j1) coded by Sequence ID No. 75 CDRH1 composed of the amino acid sequence of SEQ ID NO: 76, CDRH2 composed of the amino acid sequence of SEQ ID NO: 77, and CDRH3 composed of the amino acid sequence of SEQ ID NO: 77, as well as polynucleotides encoding the sequences The polynucleotide of CDRL1 composed of the amino acid sequence recorded in SEQ ID NO. 72, CDRL2 composed of the amino acid sequence recorded in SEQ ID NO. 73, and CDRL3 composed of the amino acid sequence recorded in SEQ ID NO. 74; (j2) A polynucleotide having at least 95% sequence identity with the nucleotide sequence described in (j1); and (j3) One to several polynucleotides described in (j1) or (j2) A polynucleotide in which nucleotides have been substituted, deleted, or added.

[57] The polynucleotide as described in [55], which includes a polynucleotide selected from any one of the groups including the following (i1) to (i3): (i1) encoding is described in Sequence ID No. 55 CDRH1 composed of the amino acid sequence, CDRH2 composed of the amino acid sequence described in SEQ ID NO. 56, and CDRH3 composed of the amino acid sequence described in SEQ ID NO. 57, and polynucleotides encoding CDRH identified by the sequence The polynucleotide of CDRL1 composed of the amino acid sequence described in SEQ ID NO. 52, CDRL2 composed of the amino acid sequence described in SEQ ID NO. 53, and CDRL3 composed of the amino acid sequence described in SEQ ID NO. 54; ( i2) A polynucleotide having at least 95% sequence identity with the nucleotide sequence described in (i1); and (i3) The polynucleotide described in (i1) or (i2), having 1 to several nuclei A polynucleotide in which nucleotides are substituted, deleted, or added.

[58] The polynucleotide described in [55], which includes a polynucleotide selected from the group consisting of (k1) to (k3) described in any one of the following: (k1) coded by Sequence ID No. 65 CDRH1 composed of the amino acid sequence of SEQ ID NO: 66, CDRH2 composed of the amino acid sequence of SEQ ID NO: 67, and CDRH3 composed of the amino acid sequence of SEQ ID NO: 67, as well as polynucleotides encoding the sequence The polynucleotide of CDRL1 composed of the amino acid sequence recorded in SEQ ID NO. 62, CDRL2 composed of the amino acid sequence recorded in SEQ ID NO. 63, and CDRL3 composed of the amino acid sequence recorded in SEQ ID NO. 64; (k2) A polynucleotide having at least 95% sequence identity with the nucleotide sequence described in (k1); and (k3) One to several polynucleotides described in (k1) or (k2) A polynucleotide in which nucleotides have been substituted, deleted, or added.

[59] The polynucleotide as described in [55], which includes a polynucleotide selected from the group consisting of any one of the following (m1) to (m3): (m1) is encoded by Sequence ID No. 85 CDRH1 composed of the amino acid sequence recorded in SEQ ID NO: 86, CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 86, and CDRH3 composed of the amino acid sequence recorded in SEQ ID NO: 87, as well as polynucleotides encoding Polynucleotide of CDRL1 composed of the amino acid sequence described in SEQ ID NO: 82, CDRL2 composed of the amino acid sequence described in SEQ ID NO: 83, and CDRL3 composed of the amino acid sequence described in SEQ ID NO: 84 ; (m2) A polynucleotide having at least 95% sequence identity with the nucleotide sequence described in (m1); and (m3) One to several polynucleotides described in (m1) or (m2) Polynucleotides in which nucleotides have been substituted, deleted or added.

[60] An expression vector containing the polynucleotide described in any one of [55] to [59].

[61] A host cell transformed by an expression vector as described in [60].

[62] A method for producing the antibody or functional fragment of the antibody as described in any one of [1] to [41], which includes culturing the host cell as described in [61], and extracting the antibody of interest from the cultured product. Steps for making functional fragments of the antibody.

[63] The antibody or functional fragment of the antibody as described in any one of [1] to [41], wherein the activation conveyed through the CD147 message is activation of p38 and/or activation of SMAD4.

[64] The antibody or functional fragment of the antibody as described in [63], wherein the activation of p38MAPK and/or the activation of SMAD4 is an increase in the expression amount of p38MAPK, phosphorylation of p38MAPK, phosphorylation of HSP27, and expression of CXCL8 Increase in amount, increase in rhoB expression, decrease in KLF5 mRNA or decrease in KLF5 protein expression.

[65] A method of treating tumors, characterized by administering the antibody described in [63] or [64] or an antigen-binding fragment of the antibody.

[66] A method of predicting response to cancer treatment, which includes using a biological sample derived from a cancer patient, detecting the expression of SMAD4 or the expression of KLF5 contained in the biological sample, and detecting the expression of SMAD4 Patients or patients whose expression of KLF5 is reduced or deleted are determined to be candidates for use of the antibody or functional fragment of the antibody as described in any one of [1] to [41] or any of [42] to [48]. A documented pharmaceutical composition is responsive to cancer treatment.

[67] A method of screening candidates for cancer treatment, which includes using a biological sample derived from a cancer patient, detecting the presence or absence of expression of SMAD4 in the biological sample or detecting the expression of KLF5, and identifying patients with detected SMAD4 or Patients whose expression of KLF5 is reduced or deleted are screened for use as antibodies or functional fragments of the antibodies described in any one of [1] to [41] or any one of [42] to [48] The medical compositions described are intended for cancer treatment.

[68] A cancer treatment method, which includes using a biological sample derived from a cancer patient, detecting the presence or absence of expression of SMAD4 contained in the biological sample or detecting the expression of KLF5, and testing the patient in which SMAD4 is detected or detecting the expression of KLF5. Patients with reduced or missing KLF5 expression should be administered the antibody or functional fragment of the antibody as described in any one of [1] to [41] or the medicine as described in any of [42] to [48] composition.

[69] A kit for determining the use of an antibody as described in any one of [1] to [41] or a functional fragment of the antibody or as described in any of [42] to [48] The response of the pharmaceutical composition to cancer treatment at least includes a means of detecting the expression of SMAD4 or the expression of KLF5 in a biological sample derived from a cancer patient.

[70] An antibody-drug complex, which is an antibody or an antigen-binding fragment of the antibody described in any one of [1] to [41] combined with other drugs.

[71] A bispecific antibody comprising an antigen-binding fragment of the antibody according to any one of [1] to [41] and an antigen-binding fragment that binds to an antigen other than CD147.

The antibody system of the present invention specifically recognizes CD147 antibodies, is characterized by activating relevant message molecules that pass through CD147, and has high anti-tumor activity. CD147 is expressed not only in tumor cells but also in blood cells. However, the antibody of the present invention has no effect on T cells and PBMC and is not dependent on effector function. Therefore, it has so-called advantages in the development of anti-tumor agents. Advantages of less security concerns. In addition, the antibody system of the present invention shows extremely high anti-tumor activity. The antibody of the present invention is bound to liver cancer cells and exhibits significantly stronger efficacy than sorafenib, one of the standard therapeutic drugs for liver cancer. The antibody of the present invention is bound to pancreatic cancer cells and exhibits a significantly stronger medicinal effect than gemcitabine used as one of the standard therapeutic drugs for pancreatic cancer. Furthermore, the antibody of the present invention is bound to chronic myelogenous leukemia cells and exhibits significantly stronger efficacy than imatinib, which is one of the standard therapeutic drugs for chronic myelogenous leukemia.

Figure 1 shows (a)~(c): changes in tumor volume in the PANC-1 xenograft model.

Figure 2-1 Binding of the system to human CD147 or monkey CD147.

Figure 2-2 shows the binding to human CD147 or monkey CD147.

Figure 2-3 shows the binding to human CD147 or monkey CD147.

Figure 3 is a sequence comparison of the amino acid sequences of human CD147 and cynomolgus monkey CD147 and the positions of mu1~mu9.

Figure 4-1 shows the nucleotide sequence and amino acid sequence of the light chain variable region of LN22R8, as well as the amino acid sequences of CDRL1, CDRL2 and CDRL3 of the LN22R8 light chain.

Figure 4-2 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of LN22R8, as well as the amino acid sequences of CDRH1, CDRH2 and CDRH3 of the LN22R8 heavy chain.

Figure 5-1 shows the nucleotide sequence and amino acid sequence of the light chain variable region of 2P10F2, as well as the amino acid sequences of CDRL1, CDRL2 and CDRL3 of the 2P10F2 light chain.

Figure 5-2 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of 2P10F2, and the amino acid sequences of CDRH1, CDRH2 and CDRH3 of the 2P10F2 heavy chain.

Figure 6 shows the ADCC activity of human chimeric antibodies.

Figure 7 shows the CDC activity of human chimeric antibodies.

Figure 8 shows the ADCP activity of human chimeric antibodies.

Figure 9-1 Lines (a) to (d): Anti-tumor effects of each antibody in the MIA PaCa-2 subcutaneous transplantation model using NOD-scid mice.

Figure 9-2 Lines (e) to (g): Anti-tumor effects of each antibody in the MIA PaCa-2 subcutaneous transplantation model using NOD-scid mice.

Figure 10 shows the anti-tumor effect of LN22R8chIgG4P in the MIA PaCa-2 subcutaneous transplantation model using NOG mice.

Figure 11 Lines (a) to (d): anti-tumor effects of r#84, r#101, r#110 or r#131 in the MIA PaCa-2 subcutaneous transplantation model using NOD-scid mice.

Figure 12 is a competition assay between 2P10F2 and r#84, r#101, r#110 or r#131.

Figure 13-1 (a)~(d): Anti-tumor effects of each chimeric antibody in the MIA PaCa-2 subcutaneous transplantation model.

Figure 13-2 (e) ~ (h): anti-tumor effects of each chimeric antibody in the MIA PaCa-2 subcutaneous transplantation model.

Figure 13-3 (i)~(l): Anti-tumor effects of each chimeric antibody in the MIA PaCa-2 subcutaneous transplantation model.

Figure 13-4 (m) and (n): anti-tumor effects of each chimeric antibody in the MIA PaCa-2 subcutaneous transplantation model.

Figure 14 shows the design of each variable region of (a) humanized antibody heavy chain #84H1h and (b) humanized antibody light chain #84L2h.

Figure 15 shows the design of each variable region of (a) humanized antibody heavy chain #101H1h and (b) humanized antibody light chain #101L2h.

Figure 16 shows (a) humanized antibody heavy chain #110H1h, (b) humanized antibody heavy chain #110H13h, (c) humanized antibody light chain #110L4h, (d) humanized antibody light chain #110L2h and (e) Design of variable regions of humanized antibody light chain #110L12h.

Figure 17 shows the design of each variable region of (a) humanized antibody heavy chain #131H2h and (b) humanized antibody light chain #131L2h.

Figure 18 (a) ~ (d): Anti-tumor effects of various humanized antibodies in the MIA PaCa-2 subcutaneous transplantation model

Figure 19 shows p38 MAPK phosphorylation by anti-human CD147 human chimeric antibodies.

Figure 20 shows (a) and (b): phosphorylation of HSP27 by anti-human CD147 human chimeric antibody.

Figure 21 (a) ~ (c): p38MAPK phosphorylation in tumors caused by anti-human CD147 human chimeric antibodies.

Figure 22 shows (a) to (c): the expression of cxc18 and rhoB caused by anti-human CD147 human chimeric antibodies.

Figure 23 shows (a) to (c): the expression of cxcl8 and rhoB caused by the formation of anti-human CD147 rat antibody and its human chimeric antibody.

Figure 24 shows the results of evaluating the anti-tumor effects of anti-CD147 antibodies and gemcitabine using SMAD4-negative pancreatic cancer cells BxPC-3.

Figure 25 shows (a) to (c): the results of evaluating the anti-tumor effect of anti-CD147 human chimeric antibodies using BxPC-3 expressing SMAD4.

Figure 26 (a) and (b): Evaluation of phosphorylation of p38MAPK when an anti-CD147 human chimeric antibody was administered to BxPC-3 pancreatic cancer cells expressing SMAD4.

Figure 27 shows the anti-tumor effect of anti-CD147 human chimeric antibody in gemcitabine-resistant pancreatic cancer tumor model.

Figure 28 shows (a) confirmation of the expression of CD147 in Hep G2 cells using flow cytometry; (b) activation of p38MAPK by humanized CD147 antibodies in liver cancer cells.

Figure 29 (a) ~ (d): Comparison of the anti-tumor effects of CD147 antibodies and sorafenib in liver cancer.

Figure 30 shows the binding of CD147-APC in CD3 and CD4 positive cells and CD3 and CD8 positive cells.

Figure 31 is an evaluation of the effect of anti-human CD147 antibodies on inducing proliferation of human peripheral blood mononuclear cells.

Figure 32 is an evaluation of the effect of anti-human CD147 antibodies on interleukin production in human peripheral blood lymphocytes.

Figure 33-1 shows the nucleotide sequence and amino acid sequence of the light chain variable region of rat_CD147_#84, and the amino acid sequence of CDRL1, CDRL2 and CDRL3 of the light chain of rat_CD147_#84.

Figure 33-2 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of rat_CD147_#84, and the amino acid sequence of CDRH1, CDRH2 and CDRH3 of the heavy chain of rat_CD147_#84.

Figure 34-1 shows the nucleotide sequence and amino acid sequence of the light chain variable region of rat_CD147_#101, and the amino acid sequence of CDRL1, CDRL2 and CDRL3 of the light chain of rat_CD147_#101.

Figure 34-2 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of rat_CD147_#101, and the amino acid sequence of CDRH1, CDRH2 and CDRH3 of the heavy chain of rat_CD147_#101.

Figure 35-1 shows the nucleotide sequence and amino acid sequence of the light chain variable region of rat_CD147_#110, and the amino acid sequence of CDRL1, CDRL2 and CDRL3 of the light chain of rat_CD147_#110.

Figure 35-2 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of rat_CD147_#110, and the amino acid sequence of CDRH1, CDRH2 and CDRH3 of the heavy chain of rat_CD147_#110.

Figure 36-1 shows the nucleotide sequence and amino acid sequence of the light chain variable region of rat_CD147_#131, and the amino acid sequence of CDRL1, CDRL2 and CDRL3 of the light chain of rat_CD147_#131.

Figure 36-2 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of rat_CD147_#131, and the amino acid sequence of CDRH1, CDRH2 and CDRH3 of the heavy chain of rat_CD147_#131.

Figure 37-1 shows the amino acid sequence and nucleotide sequence of #84H1hIgG2.

Figure 37-2 shows the amino acid sequence and nucleotide sequence of #84H1hIgG4P.

Figure 37-3 shows the amino acid sequence and nucleotide sequence of #84L2h.

Figure 38-1 shows the amino acid sequence and nucleotide sequence of #101H1hIgG2.

Figure 38-2 shows the amino acid sequence and nucleotide sequence of #101H1hIgG4P.

Figure 38-3 shows the amino acid sequence and nucleotide sequence of #101L2h.

Figure 39-1 shows the amino acid sequence and nucleotide sequence of #110H1hIgG4P.

Figure 39-2 shows the amino acid sequence and nucleotide sequence of #110H13hIgG4P.

Figure 39-3 shows the amino acid sequence and nucleotide sequence of #110L4h.

Figure 39-4 shows the amino acid sequence and nucleotide sequence of #110L2h.

Figure 39-5 shows the amino acid sequence and nucleotide sequence of #110L12h.

Figure 40-1 shows the amino acid sequence and nucleotide sequence of #131H2hIgG2.

Figure 40-2 shows the amino acid sequence and nucleotide sequence of #131L2h.

Figure 41 is a strip diagram showing two complexes included in the asymmetric unit. CD147 is shown in black, and the antibody heavy chain (H CHAIN) and light chain (L CHAIN) are shown in gray.

Figure 42 shows the interaction surface between CD147 and antibodies. The CD147 amino acids near the antibody are represented as stick models and labeled with text. Other parts of CD147 are modeled with black ribbons. On the other hand, the amino acids of the antibody near CD147 are represented by a thin line model, and other parts are represented by a gray ribbon model.

Figure 43 shows the anti-tumor effects of chimeric and humanized CD147 antibodies in gastric cancer models.

Figure 44 shows the anti-tumor effect of humanized CD147 antibody in chronic myelogenous leukemia (CML) model.

Figure 45 shows the anti-tumor effect of humanized CD147 antibody in colorectal cancer model.

Figure 46 shows the anti-tumor effect of humanized CD147 antibody in renal cancer model.

Figure 47 shows the anti-tumor effect of humanized CD147 antibody in acute myeloid leukemia (AML) model.

Figure 48 shows the anti-tumor effect of humanized CD147 antibody in pancreatic cancer model.

Figure 49 shows the results of competing ELISA.

Figure 50 is a comparison of the anti-tumor effects of (a) MIA PaCa-2 cells and (b) humanized CD147 antibodies using MIA PaCa-2 cells expressing KLF5.

Form used to implement the invention

Hereinafter, suitable modes for implementing the present invention will be described with reference to the drawings. In addition, the embodiment described below is an example of a representative embodiment of the present invention, and the scope of the present invention is not interpreted narrowly by this example.

(definition)

In this specification, "cancer" and "tumor" are used with the same meaning, and each is used when intended to refer to solid cancer, non-solid cancer, or both unless otherwise specified.

In this specification, the term "gene" includes not only DNA but also its mRNA, cDNA, and cRNA.

In this specification, "polynucleotide" or "nucleotide" is used in the same sense as nucleic acid, and also includes DNA, RNA, probes, oligonucleotides, and primers.

In this specification, "polypeptide" and "protein" are used without distinction.

In this specification, "cells" also include cells in individual animals and cultured cells.

In this specification, "CD147" is used in the same sense as CD147 protein.

In this specification, "functional fragment of an antibody" is also called "antigen-binding fragment of an antibody", which means a partial fragment of an antibody that has binding activity to an antigen, including Fab, F(ab')2, Fv, scFv, Diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments, etc. Furthermore, Fab', which is a monovalent fragment of the variable region of an antibody in which F(ab')2 has been treated under reducing conditions, is also included in the antigen-binding fragments of antibodies. However, it is not limited to these molecules as long as it has the ability to bind to antigens. In addition, these antigen-binding fragments include not only those in which the full-length molecule of the antibody protein has been treated with appropriate enzymes, but also include proteins produced in appropriate host cells using genetically engineered antibody genes.

In this specification, "effector activity" refers to antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, or antibody Any one or more of Antibody-dependent cellular phagocytosis (hereinafter, ADCP) activity.

In this specification, "effector function" means that each "effector activity" is exerted.

Antibody-dependent cellular cytotoxicity (ADCC) activity can be measured by a ⁵¹ Cr release assay that determines cell death resulting from contact between immune cells with effector activity, antibodies, and target cells labeled with ⁵¹ Cr. The ADCC activity of the human CD147 antibody of the invention was determined as follows. To evaluate the ADCC activity of human CD147 antibodies, human peripheral blood mononuclear cells (PBMC) were used as effector cells, and CD147-positive human cancer cell lines (for example, pancreatic cancer line MIA PaCa-2) were used as ADCC target cells. . Cancer cells labeled with the radioactive isotope ⁵¹ Cr and the antibody of the evaluation object were treated at a concentration of 0.5 or 5 μg/ml at 4°C for 30 minutes, and then PBMC isolated from human peripheral blood were added at a ratio of 20 times that of the cancer cells. , incubate for 4 hours at 37 degrees Celsius in the presence of 5% CO2. TopCount NXT v2.53 was used to measure ^{the 51} Cr released in the supernatant and obtain the total release value. The measured value of ⁵¹ Cr released by treating ⁵¹ Cr-labeled cancer cells with Triton-100 was set as the maximum release value, and the measured value of ⁵¹ Cr released by treatment of antibody-treated cells without adding PBMC was set as the maximum release value. For the spontaneous release value, the specific release % is calculated from the following formula. As a negative control sample, human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was used. The measurement system was performed three times, and the average value and standard deviation were calculated.

Specific release % = (total release - spontaneous release) maximum release

Complement-dependent cytotoxicity (CDC) activity can be evaluated by measuring cell death resulting from contact between complement, antibodies and target cells contained in the blood. The CDC activity of the human CD147 antibody of the present invention was determined as follows. The complement-dependent cytocidal activity (CDC activity) of the human CD147 antibody to be evaluated was evaluated using the human pancreatic cancer strain MIA PaCa-2 as a target cell. Commercially available rabbit complement (Low Tox-M Rabbit Complement, CEDARLANE LABORATORIES LIMITED, Cat. CL3051) was used as complement. Human IgG (hlgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was used as a negative control antibody for CDC activity. The antibodies to be evaluated and the negative target antibodies were each treated at a concentration of 0, 0.1, 1, or 10 μg/ml at 4°C for 1 hour, then rabbit complement was added to make the final concentration 7.5%, and the solution was maintained at 37°C and 5% CO2. After heating for 3 hours, the intracellular ATP contained in the living cells was measured using CellTiter-Glo Lumimescent Cell Viability Assay (Promega, Cat. G7572). The luminescence signal obtained using CellTiter-Glo Lumimescent Cell Viability Assay was quantified using EnVision 2104 Multilabel Reader (PerkinElmr). The measurement was performed three times, and the average value and standard deviation were calculated. The luminescence signal obtained from untreated cells was set as 100%, and the antibody- and complement-dependent decrease in luminescence signal was regarded as CDC activity.

Antibody-dependent cellular phagocytosis (ADCP) activity can be measured using a double fluorescent labeling method by phagocytosis that occurs by bringing phagocytic immune cells, antibodies, and target cells into contact.

The ADCP activity of the human CD147 antibody of the present invention was measured in the following manner. It has been reported that human IgG antibodies exhibit cytocidal activity against cancer cells by inducing antibody-dependent monocyte-macrophage phagocytosis (ADCP) through interaction with mouse Fcγ receptors ( Overdijk et al., Journal of Immunology, 1-9, 2012). The ADCP activity of the human chimeric antibody of the present invention was evaluated using RAW264.7 (ATCC, TIB-71) as effector cells and human pancreatic cancer strain PANC-1 or MIA PaCa-2 as ADCP target cells. The ADCP-labeled cells labeled with PKH67 Green Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (SIGMA, Cat.MINI67-1KIT) and the evaluated antibodies were treated with the evaluated antibodies at a concentration of 20 μg/ml at 4°C for 1 hour. , add RAW264.7 cells labeled with PKH26 Red Fluorescent Cell Linker Lit for General Cell Membrane Labeling (SIGMA, Cat. PKH26GL-1KT) at 5 times the amount of ADCP-labeled cells, and heat for 3 hours at 37°C in the presence of 5% CO2 hours. A flow cytometer (BD, CantoII) was used to determine the rate of PKH26-positive cells that migrated to PKH67 signal-positive cells through phagocytosis. As a negative control sample, a sample of treated human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was measured in the same manner. The measurement system was performed three times, and the average value and standard deviation were calculated.

In this specification, "substantially no effector activity" or "effector activity is reduced or missing" means that the antibody system does not show activity in at least one of ADCC activity, CDC activity or ADCP activity, or is lower than that. Activity to the extent that its functions are not fully utilized. "Substantially no effector activity" or "effector activity is reduced or missing" means, for example, that in the above activity evaluation method, the activity of the antibody evaluated is the same level as that of the negative control.

In this specification, "the ADCC activity is reduced or missing" means that the evaluated antibodies do not show ADCC activity, or the activity is so low that their functions are not fully exerted. "The ADCC activity is reduced or missing" means, for example, that in the above-mentioned activity evaluation method, the activity of the antibody evaluated is the same level as that of the negative control.

In this specification, "CDC activity is reduced or missing" means that the evaluated antibodies do not show CDC activity, or the activity is so low that their functions are not fully exerted. "CDC activity is reduced or missing" means that, for example, in the above-mentioned activity evaluation method, the activity of the antibody evaluated is the same level as that of the negative control.

In this specification, "the ADCP activity is reduced or missing" means that the evaluated antibodies do not show ADCP activity, or the activity is so low that their functions are not fully exerted. "The ADCP activity is reduced or absent" means, for example, that in the above-mentioned activity evaluation method, the activity of the antibody evaluated is the same level as that of the negative control.

"ADCC activity is reduced or absent", "CDC activity is reduced or absent" or "ADCP activity is reduced or absent" means, for example, that in the respective above-mentioned activity evaluation methods, the activity of the antibody evaluated is the same as that of the negative control degree of activity.

In this specification, "activation of message transmission through CD147", "activation of related message molecules through CD147", "activation of CD147" or "activation of CD147" refers to the cell message transmission system through CD147 Activation means activating at least one of the downstream related message molecules of CD147. Activation transmitted through the CD147 message means that the expression of genes downstream of the CD147 message is increased or decreased, or the expression of the protein is increased or decreased, or the phosphorylation of the protein is increased or decreased. Examples of related signaling molecules downstream of CD147 include FAK, MEK, Erk, JAK/STAT, AKT, or MAP kinase (MAPK), or activation of these further downstream signaling molecules. Examples of MAPK include ERK1/2, JNK, and p38MAPK, and p38MAPK is more preferred. As for signaling molecules further downstream of MAPK, for example, HSP27, cxc18 or SMAD (for example, SMAD2, SMAD3 and/or SMAD4) can be cited. "Activation of CD147" includes, for example, an increase in the expression level of p38MAPK mRNA, an increase in the amount of protein expression of p38MAPK, phosphorylation of p38MAPK, and phosphorylation of HSP27 (for example, phosphorylation of Ser82 of HSP27 or HSP27 Phosphorylation of Ser15), increase in cxcl8 mRNA expression, increase in cxcl8 protein expression, increase in rhoB mRNA expression or increase in rhoB protein expression through SMAD message activation, or decrease in KLF5 mRNA or KLF5 protein Reduction in performance.

In this specification, "antitope" means part of the peptide or part of the three-dimensional structure of CD147 that a specific anti-CD147 antibody binds to. The epitope of the partial peptide of CD147 can be determined by immunoassay and other methods well known to those skilled in the art. First, various partial structures of the antigen are made. For the preparation of partial constructs, well-known oligonucleotide synthesis techniques can be used. For example, using gene recombination technology well known to those skilled in the art, a series of polypeptides sequentially shortened to appropriate lengths from the C-terminal or N-terminal of CD147 are produced, and then the reactivity of the antibody to them is examined, and After the rough identification site is determined, shorter peptides are synthesized and the reactivity with these peptides is examined to determine the epitope. Furthermore, in the case where an antibody that binds to a membrane protein containing multiple extracellular functional domains uses a three-dimensional structure containing multiple functional domains as an epitope, the amino acid sequence of a specific extracellular functional domain can be changed. Change the three-dimensional structure to determine which functional domain to combine with. The epitope, which is the partial three-dimensional structure of the antigen to which a specific antibody binds, can also be determined by using X-ray structural analysis to identify the amino acid residues of the antigen adjacent to the aforementioned antibody.

If the second antibody binds to part of the peptide or part of the three-dimensional structure that the first antibody binds to, it can be determined that the first antibody and the second antibody have a common epitope. Furthermore, by confirming that the second antibody is cross-competitive for the binding of the first antibody to the antigen (i.e., the second antibody will hinder the binding of the first antibody to the antigen), even if the sequence or structure of the specific epitope is not determined , it can also be determined that the first antibody and the second antibody bind to the same epitope. If the first antibody and the second antibody bind to the same epitope, and the first antibody has special effects such as anti-tumor activity, it can be expected that the second antibody will also have the same activity.

It is known that the heavy chain and light chain of antibody molecules each have three complementarity determining regions (CDRs). The complementarity determining region, also known as the hypervariable domain, is located within the variable regions of the heavy and light chains of the antibody. It is a particularly highly variable part of the primary structure of the polypeptides of the heavy and light chains. The primary structure of the chain is separated into three places. In this specification, regarding the complementarity-determining regions of antibodies, the complementarity-determining regions of the heavy chain are labeled CDRH1, CDRH2, and CDRH3 from the amino terminal side of the amino acid sequence of the heavy chain, and the complementarity-determining regions of the light chain are labeled CDRH1, CDRH2, and CDRH3. The amino terminal side labels of the chain amino acid sequences are CDRL1, CDRL2, and CDRL3. These parts are close to each other in three-dimensional structure, which determines the specificity for the bound antigen.

In this specification, when "1 to several" and "1 or several" are described, "several" means 2 to 10. The number is preferably 10 or less, more preferably 5 or 6 or less, and still more preferably 2 or 3.

(CD147)

CD147 is known to be a primary transmembrane protein with 2 to 3 immunoglobulin-like domains. Through the interaction of CD147 with each other, it interacts with CD44, integrin family molecules, CD98, VEGFR, CypA/B, and MCT1/3. /4 etc. will participate in extracellular and cell membrane surface molecular interactions of proliferation, infiltration, inflammation, and activate downstream message-related molecules, FAK, MEK, Erk, JAK/STAT, AKT, and MAPK family molecules, which will promote the activation of MMP The production of proteases, cancer proliferation, metastasis, and invasion.

There are three known variants of human CD147. Variant 1 is a primary transmembrane protein that is specifically expressed in the omentum and has three immunoglobulin-like domains (each in this specification, sometimes referred to as D0, D1 and D2); variant 2 is expressed in T cells Or a primary transmembrane protein that is expressed in various normal cells and has two immunoglobulin-like domains (D1, D2) that are reported to be increased in various cancer tissues; variant 3 has A primary transmembrane protein with an immunoglobulin-like domain.

The amino acid sequence and nucleotide sequence of variant 1 of human CD147 can be obtained by referring to GenBank accession numbers NP_001719.2 and NM_001728.3. In this specification, the amino acid sequence is also disclosed as sequence identification number 1, and the nucleic acid sequence is The nucleotide sequence is also disclosed as SEQ ID NO: 2. The three immunoglobulin-like domains of variant 1, in terms of sequence identification number 1, are amino acid numbers 22 to 138 (D0), amino acid numbers 140 to 218 (D1), and amino acid number 223. ~323(D2) (Redzic, J., J. Mol. Biol., 2011, 68-82) (Grass et al., Biosol. Rep, 2016, 1-16). In addition, the membrane-penetrating region of variant 1 has amino acid numbers 324 to 344 in terms of sequence identification number 1.

The amino acid sequence and nucleotide sequence of variant 2 of human CD147 can be obtained by referring to the GenBank accession numbers NP_940991.1 and NM_198589.2. In this specification, the amino acid sequence is also disclosed as sequence identification number 3, and the nucleic acid sequence is The nucleotide sequence is disclosed as SEQ ID NO: 4. The two immunoglobulin-like domains (D1 and D2) of variant 2, as far as sequence identification number 3 is concerned, are amino acid numbers 24 to 102 (D1) and amino acid numbers 107 to 207 (D2) respectively. In addition, the membrane-penetrating region of variant 2 has amino acid numbers 208 to 228 in terms of sequence identification number 3 (Grass et al., Biosol. Rep, 2016, 1-16).

The amino acid sequence and nucleotide sequence of variant 3 of human CD147 were obtained by referring to GenBank accession numbers NP_940992.1 and NM_198590.2. In addition, the human CD147 gene can also be obtained from commercial sources.

The amino acid sequence and nucleotide sequence of cynomolgus monkey CD147 (also described as monkey CD147 in this specification) can be obtained by referring to GenBank accession numbers XP_005587354.1 and XM_005587297.1. In addition, the monkey CD147 gene can also be obtained from commercial sources. The amino acid sequence and nucleotide sequence of mouse CD147 can be obtained by referring to GenBank accession numbers NP_001070652.1 and NM_001077184.1. In addition, the mouse CD147 gene can also be obtained from commercial sources.

CD147 used in the present invention can be synthesized in vitro, or can be produced in host cells through genetic manipulation. Specifically, it can be synthesized by incorporating CD147 cDNA into an expressible vector in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or by using other prokaryotic or eukaryotic host cells. Transformed to express CD147, the protein is obtained.

CD147 cDNA can be obtained by, for example, using a cDNA library expressing CD147 cDNA as a template and using primers that specifically amplify CD147 cDNA to perform polymerase chain reaction (hereinafter referred to as "PCR") (Saiki, R.K., et al., Science, (1988) 239, 487-49) by the so-called PCR method.

Also, hybridize under stringent conditions with a polynucleotide comprising a nucleotide sequence that is complementary to the nucleotide sequence encoding human, monkey or mouse CD147, and encode a polynucleoside that has a biological activity equivalent to CD147. Acid is also contained in the cDNA of CD147. Furthermore, a splicing variant (splicing variant) transcribed from the human, monkey or mouse CD147 locus or a polynucleotide hybridizing thereto under stringent conditions and encoding a protein having the same biological activity as CD147 , also included in the cDNA of CD147.

In addition, one or more amino acids in the amino acid sequence of human, monkey or mouse CD147, or the amino acid sequence in which the message sequence has been removed, have been substituted, deleted, or added. Proteins composed of amino acid sequences and having the same biological activity as CD147 are also included in CD147. Furthermore, the amino acid sequence encoded by the splice variant transcribed from the human, monkey or mouse CD147 locus, or one or several amino acids in the amino acid sequence are substituted, deleted, or Proteins composed of added amino acid sequences and having the same biological activity as CD147 are also included in CD147.

(Production of anti-CD147 antibody)

The anti-CD147 antibody of the present invention is obtained by immunizing non-human animals with the target antigen. After the immunization is established, lymph fluid, lymphoid tissue, blood cell samples or cells derived from bone marrow are collected from the animals, and the method is followed by well-known methods (for example, Kohler and Milstein , Nature (1975) 256, p.495-497, Kennet, R.ed., Monoclonal Antibodies, p.365-367, Plenum Press, N.Y. (1980)), allowing antibody-producing cells that produce anti-CD147 antibodies to interact with bone marrow Tumor cells are fused to create fusion tumors and monoclonal antibodies can be obtained. Specific examples of this method are described in WO2009/48072 (published on April 16, 2009) and WO2010/117011 (published on October 14, 2010). Examples of monoclonal antibodies obtained in this manner include LN22R8, 2P10F2, rat_CD147_#84, rat_CD147_#101, rat_CD147_#110, and rat_CD147_#131. However, the method of obtaining monoclonal antibodies is an established field and is not limited to the specific examples mentioned above.

The antibodies of the present invention, in addition to the above-mentioned anti-CD147 monoclonal antibodies, also include genetically recombinant antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies and humanized antibodies. ) antibodies, human antibodies, etc. Such antibodies can be produced using known methods.

Examples of chimeric antibodies include antibodies in which the variable region and the constant region of the antibody are heterologous to each other. For example, a chimeric antibody is one in which the variable region of a mouse- or rat-derived antibody is joined to a human-derived constant region. Antibodies (see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)). An example of a chimeric antibody derived from LN22R8 is an antibody having an amino acid sequence consisting of amino acid residues No. 20 to No. 471 of Sequence ID No. 33 in the sequence listing. chain, a heavy chain having an amino acid sequence consisting of amino acid residues Nos. 20 to 467 of SEQ ID NO: 35 or a heavy chain having an amino acid sequence composed of amino acid residues Nos. 20 to 468 of SEQ ID NO: 37 A heavy chain composed of an amino acid sequence, and a light chain having an amino acid sequence composed of amino acid residues Nos. 21 to 234 of SEQ ID NO: 31. An example of a chimeric antibody derived from 2P10F2 is an antibody having an amino acid sequence consisting of amino acid residues Nos. 20 to 466 of SEQ ID NO: 43 in the sequence listing. chain, a heavy chain having an amino acid sequence consisting of amino acid residues No. 20 to 462 of Sequence ID No. 45 in the Sequence Listing, or a heavy chain having amino acid residues Nos. 20 to 463 of Sequence ID No. 47 A heavy chain having an amino acid sequence composed of amino acid residues, and a light chain having an amino acid sequence composed of amino acid residues Nos. 21 to 234 of SEQ ID NO: 41.

An example of a chimeric antibody derived from rat_CD147_#84 is an antibody having an amino acid sequence consisting of amino acid residues No. 20 to No. 470 of Sequence ID No. 92 in the Sequence Listing. A heavy chain having an amino acid sequence consisting of amino acid residues No. 20 to 466 of Sequence ID No. 94 in the Sequence Listing, and a heavy chain having an amino acid sequence consisting of Amino Acid Residues No. 20 to 467 of Sequence ID No. 96 A heavy chain with an amino acid sequence composed of amino acid residues, a heavy chain with an amino acid sequence composed of amino acid residues No. 20 to 470 of SEQ ID NO: 98, or a heavy chain having an amino acid sequence composed of SEQ ID NO: 100 A heavy chain having an amino acid sequence composed of amino acid residues No. 20 to 467, and a heavy chain having an amino acid sequence composed of amino acid residues Nos. 21 to 234 of SEQ ID NO: 90 light chain.

An example of the chimeric antibody derived from rat_CD147_#101 is an antibody having an amino acid sequence consisting of amino acid residues No. 20 to No. 463 of Sequence ID No. 104 in the Sequence Listing. A heavy chain having an amino acid sequence consisting of amino acid residues No. 20 to 464 of Sequence ID No. 106 in the Sequence Listing or a heavy chain having an amine sequence consisting of Nos. 20 to 464 of Sequence ID No. 108 The heavy chain has an amino acid sequence composed of amino acid residues, and the light chain has an amino acid sequence composed of amino acid residues No. 21 to 234 of SEQ ID NO: 102.

An example of a chimeric antibody derived from rat_CD147_#110 is an antibody having an amino acid sequence consisting of amino acid residues No. 20 to No. 462 of Sequence ID No. 112 in the Sequence Listing. A heavy chain having an amino acid sequence consisting of amino acid residues No. 20 to 463 of Sequence ID No. 114 in the Sequence Listing or a heavy chain having an amine consisting of Nos. 20 to 463 of Sequence ID No. 116 The heavy chain has an amino acid sequence composed of amino acid residues, and the light chain has an amino acid sequence composed of amino acid residues No. 21 to 234 of SEQ ID NO: 110.

An example of a chimeric antibody derived from rat_CD147_#131 is an antibody having an amino acid sequence consisting of amino acid residues No. 20 to No. 464 of Sequence ID No. 120 in the Sequence Listing. The heavy chain or the heavy chain having an amino acid sequence consisting of amino acid residues No. 20 to 465 of SEQ ID NO: 122, and a heavy chain having the amino acid sequence No. 21 to 234 of SEQ ID NO: 118 The light chain of the amino acid sequence composed of residues.

Examples of humanized antibodies include antibodies in which only CDRs are incorporated into human-derived antibodies (see Nature (1986) 321, p. 522-525), and antibodies in which a portion of the framework is added in addition to the sequence of CDRs using the CDR transplantation method. The amino acid residues are also grafted into antibodies of human antibodies (International Publication No. WO90/07861).

As for the humanized antibody derived from rat_CD147_#84 antibody, as long as it retains all six CDR sequences of rat_CD147_#84, has binding activity to CD147, and activates CD147, it is an antibody of the present invention. Include. Furthermore, the heavy chain variable region of the rat_CD147_#84 antibody retains CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 55, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 56, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 57. Furthermore, the light chain variable region of rat_CD147_#84 antibody retains CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 52, CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 53, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 54. The amino acid sequence and nucleotide sequence of the light chain variable region or heavy chain variable region of the rat_CD147_#84 antibody, and the amino acid sequence of the CDR are also shown in Figure 33-1 and Figure 33-2.

Humanized antibodies derived from the rat_CD147_#101 antibody are included in the antibodies of the present invention as long as they retain all six CDR sequences of rat_CD147_#101, have binding activity to CD147, and activate CD147. Furthermore, the heavy chain variable region of rat_CD147_#101 antibody retains CDRH1 composed of the amino acid sequence represented by SEQ ID NO: 65, CDRH2 composed of the amino acid sequence represented by SEQ ID NO: 66, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 67. Furthermore, the light chain variable region of rat_CD147_#101 antibody retains CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 62, CDRL2 composed of the amino acid sequence represented by SEQ ID NO: 63, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 64. The amino acid sequence and nucleotide sequence of the light chain variable region or heavy chain variable region of the rat_CD147_#101 antibody, and the amino acid sequence of the CDR are also shown in Figure 34-1 and Figure 34-2.

Humanized antibodies derived from the rat_CD147_#110 antibody are included in the antibodies of the present invention as long as they retain all six CDR sequences of rat_CD147_#110, have binding activity to CD147, and activate CD147. Furthermore, the heavy chain variable region of the rat_CD147_#110 antibody retains CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 75, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 76, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 77. Furthermore, the light chain variable region of rat_CD147_#110 antibody retains CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 72, CDRL2 composed of the amino acid sequence represented by SEQ ID NO: 73, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 74. The amino acid sequence and nucleotide sequence of the light chain variable region or heavy chain variable region of the rat_CD147_#110 antibody, and the amino acid sequence of the CDR are also shown in Figure 35-1 and Figure 35-2.

Humanized antibodies derived from the rat_CD147_#131 antibody are included in the antibodies of the present invention as long as they retain all six CDR sequences of rat_CD147_#131, have binding activity to CD147, and activate CD147. Furthermore, the heavy chain variable region of the rat_CD147_#131 antibody retains CDRH1 composed of the amino acid sequence represented by SEQ ID NO: 85, CDRH2 composed of the amino acid sequence represented by SEQ ID NO: 86, and CDRH3 composed of the amino acid sequence shown in SEQ ID NO: 87. Furthermore, the light chain variable region of the rat_CD147_#131 antibody retains CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 82, CDRL2 composed of the amino acid sequence represented by SEQ ID NO: 83, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 84. The amino acid sequence and nucleotide sequence of the light chain variable region or heavy chain variable region of the rat_CD147_#131 antibody, and the amino acid sequence of the CDR are also shown in Figure 36-1 and Figure 36-2.

Furthermore, CDR-modified humanized antibodies in which 1 to 3 amino acid residues in each CDR are substituted with other amino acid residues can also be used as long as they have binding activity to CD147 and activate CD147. Antibodies are included in the antibodies of the present invention.

As for the humanized antibody derived from the rat_CD147_#84 antibody, human CD147 antibodies or antigen-binding fragments of the antibodies having the following (a) and (b) can be enumerated: (a) selected from the group consisting of the following (a1) to The heavy chain variable region described in any one of the groups (a4): (a1) A heavy chain consisting of amino acid residues No. 20 to 140 of the amino acid sequence represented by SEQ ID NO: 123 Variable region; (a2) A heavy chain variable region composed of amino acid residues No. 20 to 140 of the amino acid sequence represented by SEQ ID NO: 125; (a3) in (a1) or (a2) ) sequence, an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (a4) each of the sequences described in any one of (a1) to (a3) Sequences of framework regions other than CDR sequences, amino acid sequences in which one or several amino acids are deleted, substituted or added, and (b) selected from the group including the following (b1) to (b3) The light chain variable region described in any of the following: (b1) A light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 127; (b2) In the sequence of (b1), the amino acid sequence has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (b3) Each CDR in the sequence of (b1) or (b2) A sequence outside the framework region, an amino acid sequence in which one or several amino acids have been deleted, substituted or added.

Preferable examples of humanized antibodies derived from the rat_CD147_#84 antibody include a heavy chain composed of amino acid residues No. 20 to No. 140 of the amino acid sequence represented by SEQ ID NO: 125. Variable region and an antibody of a light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 127; or an antibody containing an amine represented by SEQ ID NO: 123 The heavy chain variable region composed of amino acid residues No. 20 to 140 of the amino acid sequence and the amino acid residues No. 21 to 128 of the amino acid sequence shown in Sequence ID No. 127 Antibodies with light chain variable regions.

More preferred examples of the humanized antibody derived from the rat_CD147_#84 antibody include a heavy chain composed of amino acid residues No. 20 to No. 467 of the amino acid sequence represented by SEQ ID NO: 125. And an antibody with a light chain composed of amino acid residues 21 to 234 of the amino acid sequence shown in SEQ ID NO: 127; or an antibody containing the 20th amino acid residue of the amino acid sequence shown in SEQ ID NO: 123 An antibody having a heavy chain composed of amino acid residues No. 466 and a light chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence shown in SEQ ID NO: 127.

As for the humanized antibody derived from the rat_CD147_#101 antibody, human CD147 antibodies or antigen-binding fragments of the antibodies having the following (e) and (f) can be exemplified: (e) selected from the group consisting of the following (e1) to The heavy chain variable region described in any one of the groups (e4): (e1) A heavy chain consisting of amino acid residues Nos. 20 to 137 of the amino acid sequence represented by SEQ ID NO: 129 Variable region; (e2) A heavy chain variable region composed of amino acid residues Nos. 20 to 137 of the amino acid sequence shown in SEQ ID NO: 131; (e3) in (e1) or (e2) ) sequence, an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (e4) in the sequence described in any one of (e1) to (e3) The sequence of the framework region other than each CDR sequence, the amino acid sequence in which one or several amino acids are deleted, substituted or added, and (f) is selected from the group including the following (f1) to (f3) The light chain variable region described in any one of: (f1) a light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 133; (f2 ) in the sequence of (f1), an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (f3) in each of the sequences of (f1) or (f2) The sequence of the framework region other than the CDR sequence is an amino acid sequence in which one or several amino acids are deleted, substituted or added.

Preferred examples of humanized antibodies derived from the rat_CD147_#101 antibody include a heavy chain composed of amino acid residues Nos. 20 to 137 of the amino acid sequence shown in SEQ ID NO: 129. Antibodies with variable regions and light chain variable regions composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 133, or amines represented by SEQ ID NO: 131 The heavy chain variable region composed of amino acid residues No. 20 to 137 of the amino acid sequence and the amino acid residues No. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 133 Antibodies with light chain variable regions.

More preferred examples of the humanized antibody derived from the rat_CD147_#101 antibody include a heavy chain composed of amino acid residues No. 20 to No. 463 of the amino acid sequence represented by SEQ ID NO: 129. And an antibody with a light chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence shown in SEQ ID NO: 133; or an antibody that includes No. 20 of the amino acid sequence shown in SEQ ID NO: 131 An antibody having a heavy chain composed of amino acid residues No. 464 and a light chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence shown in Sequence ID No. 133.

For humanized antibodies derived from rat_CD147_#110 antibodies, human CD147 antibodies or antigen-binding fragments of the antibodies having the following (c) and (d) can be exemplified: (c) selected from the group consisting of the following (c1)~ The heavy chain variable region described in any one of the groups of (c4): (c1) A heavy chain composed of amino acid residues Nos. 20 to 136 of the amino acid sequence represented by SEQ ID NO: 135 Variable region; (c2) A heavy chain variable region composed of amino acid residues 20 to 136 of the amino acid sequence shown in SEQ ID NO: 147; (c3) in (c1) or (c2) ), an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (c4) (recorded in any one of (c1) to (c3) The sequence of the framework region other than each CDR sequence in the sequence, the amino acid sequence in which one or several amino acids are deleted, substituted or added, and (d) is selected from the group consisting of the following (d1) ~ (d5 ) The light chain variable region described in any one of the groups: (d1) The light chain variable region consisting of amino acid residues Nos. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 137 Region; (d2) a light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO: 149; (d3) an amine represented by SEQ ID NO: 151 The light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence; (d4) The sequence described in any one of (d1) to (d3), for the framework other than each CDR sequence The sequence of the region has an amino acid sequence with at least 95% sequence identity; and (d5) the sequence of the framework region other than each CDR sequence in the sequence described in any one of (d1) to (d4), there are An amino acid sequence in which one or several amino acids are deleted, substituted or added.

Preferred examples of humanized antibodies derived from the rat_CD147_#110 antibody include a heavy chain composed of amino acid residues Nos. 20 to 136 of the amino acid sequence shown in SEQ ID NO: 135. Variable region and an antibody of a light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 137; including the amino group shown in SEQ ID NO: 147 The heavy chain variable region composed of amino acid residues No. 20 to 136 of the acid sequence and the heavy chain variable region composed of amino acid residues Nos. 21 to 128 of the amino acid sequence shown in Sequence ID No. 149 An antibody of a light chain variable region; or a heavy chain variable region composed of amino acid residues No. 20 to 136 of the amino acid sequence shown in SEQ ID NO: 147 and a heavy chain variable region shown in SEQ ID NO: 151 An antibody of the light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence.

More preferred examples of the humanized antibody derived from the rat_CD147_#110 antibody include a heavy chain composed of amino acid residues No. 20 to No. 463 of the amino acid sequence represented by SEQ ID NO: 135. And an antibody with a light chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence shown in SEQ ID NO: 137; including Nos. 20~ of the amino acid sequence shown in SEQ ID NO: 147 A heavy chain composed of amino acid residues No. 463, and a light chain composed of amino acid residues Nos. 21 to 234 of the amino acid sequence shown in Sequence ID No. 149; or an antibody containing A heavy chain composed of amino acid residues No. 20 to 463 of the amino acid sequence represented by SEQ ID NO. 147, and an amine composed of amino acid residues No. 21 to 234 of the amino acid sequence represented by SEQ ID NO. 151 Antibodies have light chains composed of amino acid residues.

As for the humanized antibody derived from the rat_CD147_#131 antibody, human CD147 antibodies or antigen-binding fragments of the antibodies having the following (g) and (h) can be exemplified: (g) is selected from the group consisting of the following (g1)~ The heavy chain variable region described in any one of the groups (g3): (g1) A heavy chain composed of amino acid residues No. 20 to No. 138 of the amino acid sequence represented by SEQ ID NO: 139 Variable region; (g2) to the sequence of (g1), an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (g3) to (g1) or (g2) ), the sequence of the framework region other than each CDR sequence in the sequence, the amino acid sequence in which one or several amino acids are deleted, substituted or added, and (h) is selected from the group consisting of the following (h1)~( The light chain variable region described in any one of the groups h3): (h1) The light chain composed of amino acid residues No. 21 to 128 of the amino acid sequence represented by Sequence ID No. 141 may variable region; (h2) the sequence in (h1), an amino acid sequence that has at least 95% sequence identity with the sequence of the framework region other than each CDR sequence; and (h3) is in (h1) or (h2) In the sequence of the framework region other than each CDR sequence, one or several amino acids are deleted, substituted or added to the amino acid sequence.

Preferred examples of humanized antibodies derived from the rat_CD147_#131 antibody include a heavy chain composed of amino acid residues Nos. 20 to 138 of the amino acid sequence shown in SEQ ID NO: 139. An antibody of a variable region and a light chain variable region composed of amino acid residues Nos. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 141.

More preferred examples of the humanized antibody derived from the rat_CD147_#131 antibody include a heavy chain composed of amino acid residues No. 20 to No. 464 of the amino acid sequence represented by SEQ ID NO: 139. And an antibody of a light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in SEQ ID NO: 141.

The humanized antibody derived from the above rat_CD147_#84 antibody, the humanized antibody derived from the rat_CD147_#101 antibody, the humanized antibody derived from the rat_CD147_#110 antibody or the humanized antibody derived from the rat_CD147_#131 antibody is preferably Activation of p38MAPK signaling through CD147 and/or SMAD4 signaling.

Further examples of the antibodies of the present invention include human antibodies. Anti-CD147 human antibodies refer to human antibodies that only have the gene sequence of antibodies derived from human chromosomes. Anti-CD147 human antibodies can be produced, for example, by a method of producing mice using human antibodies having human chromosome fragments containing genes for the heavy chain and light chain of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) 16, p.133-143; Kuroiwa, Y.et.al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H.et.al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.69-73 (Kitagawa,Y.,Matsuda,T.and Iijima,S.eds.), Kluwer Academic Publishers, 1999; Tomizuka,K.et.al., Proc.Natl.Acad.Sci .USA(2000)97, p.722-727, etc.).

Such human antibody-producing mice, specifically, can be produced by producing knockout animals and transgenic animals and mating these animals with each other to produce genetically recombinant animals. The loci of the endogenous immunoglobulin heavy chain and light chain of genetically recombinant animals are destroyed and transferred through human artificial chromosome (HAC) vector or mouse artificial chromosome (MAC) vector. The vector introduces the human immunoglobulin heavy chain and light chain loci for replacement.

In addition, through genetic recombination technology, cDNA encoding the heavy chain and light chain of the human antibody, preferably using a vector containing the cDNA, is used to transform eukaryotic cells and culture to produce the genetically recombinant human monoclonal antibody. Transformed cells whereby the antibody can also be obtained from the culture supernatant. Among them, as a host, for example, eukaryotic cells can be used, and mammalian cells such as CHO cells, lymphocytes, and myeloma are preferred.

In addition, a method of screening human antibodies derived from phage display from a human antibody library is also known (see Wormstone, I.M.et.al, Investigative Ophthalmology & Visual Science. (2002) 43(7), p.2301-2308; Carmen ,S.et.al.,Briefings in Functional Genomics and Proteomics(2002),1(2),p.189-203; Siriwardena,D.et.al.,Ophthalmology(2002)109(3),p.427 -431 etc.).

For example, a phage display method can be used in which the variable region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage and phage that bind to the antigen are selected (Nature Biotechnology (2005), 23, (9), p. 1105-1116). The DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined by analyzing the genes of the phage selected for binding to the antigen. If the DNA sequence of the scFv that binds to the antigen is known, an expression vector with the sequence can be produced and introduced into an appropriate host for expression to obtain human antibodies (WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93 /19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).

Antibodies with the same epitope as the antibodies provided by the present invention are also included in the antibodies of the present invention. Examples include antibodies having the same epitope as at least one of LN22R8, 2P10F2, rat_CD147_#84, rat_CD147_#101, rat_CD147_#110, or rat_CD147_#131.

LN22R8 and 2P10F2 of the present invention recognize the epitope represented by mu3 in Figure 3 (human CD147v1: DALPGQKTEFKVDSDDQ (sequence identification number 143), monkey CD147: DTLPGQKTDFEVDSDDL (sequence identification number 144)). The antibody of the present invention can recognize or bind to sequence identification number 143 or sequence identification number 144, or the sequence of sequence identification number 143 or sequence identification number 144 contains one or more, preferably 1 to 3, more preferably Preferably, antibodies with an amino acid sequence in which 1 or 2 amino acids are deleted, substituted or added are also included in the scope. The antibody preferably activates signaling through CD147.

Furthermore, the antibody of the present invention includes rat_CD147_#110, preferably an antibody that recognizes the same epitope as humanized #110H1L4. The antibody preferably activates signaling through CD147. The results of epitope analysis of humanized #110H1L4 are shown in Example 17.

Suitable antibodies can be selected by evaluating binding to antigens well known to those of ordinary skill in the art. The dissociation constant between the antibody and the antigen (CD147) can be measured using Biacore T200 (GE Healthcare Bioscience) which uses surface plasmon resonance (SPR) as the detection principle. For example, the binding rate constant ka1 and the dissociation rate constant can be obtained by reacting an antibody set to an appropriate concentration for an antigen immobilized as a ligand with an analyte and measuring its binding and dissociation. kd1 and dissociation definite number (KD; KD=kd1/ka1).

The evaluation of CD147 binding is not limited to the use of Biacore T200. It can also be done by machines using surface plasmon resonance (SPR) as the detection principle, and KinExA (Sapidyne) using the Kinetic Exclusion Assay as the detection principle. Instruments Company), BLItz system (Pall Company) using Bio-Layer Interferometry as the detection principle, or ELISA (Enzyme-Linked ImmunoSorbent Assay) method, etc.

As an example of other indicators when comparing the properties of antibodies, the stability of antibodies can be cited. Differential scanning calorimetry (DSC) is a method that can quickly or accurately measure the thermal denaturation midpoint (Tm) that is an excellent indicator of the relative structural stability of a protein. The difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values. It is known that the storage stability of antibodies is related to the thermal stability of antibodies to a certain extent (Lori Burton, et.al., Pharmaceutical Development and Technology (2007) 12, p. 265-273). The thermal stability can be Use it as an indicator to screen out suitable antibodies. Other indicators for screening antibodies include high yield in appropriate host cells and low agglutination in aqueous solutions. For example, because the antibody with the highest yield does not necessarily show the highest thermal stability, it is necessary to make a comprehensive judgment based on the above-mentioned indicators to select the most suitable antibody for human administration.

In addition, it is also known to use an appropriate linker to connect the full-length sequences of the heavy chain and light chain of an antibody to obtain a single chain immunoglobulin (Lee, H-S, et.al., Molecular Immunology (1999) )36, p.61-71; Shirrmann, T.et.al., mAbs (2010), 2, (1) p.1-4). This type of single-chain immunoglobulin can maintain a similar structure and activity to the original tetramer antibody through dimerization. Furthermore, the antibody of the present invention may also be an antibody having a single heavy chain variable region but no light chain sequence. This type of antibody is called a single domain antibody (sdAb) or nanobody. It has been reported that it can actually be seen in camels or llamas, and the antigen-binding ability is maintained (Muyldemans S.et.al .,Protein Eng.(1994)7(9),1129-35,Hamers-Casterman C.et.al.,Nature(1993)363(6428)446-8). The above-mentioned antibody can also be interpreted as one of the antigen-binding fragments of the antibody in the present invention.

Furthermore, the antibody-dependent cell damaging activity can be enhanced by regulating the modification of the sugar chain bound to the antibody of the present invention. Technology for regulating sugar chain modification of antibodies is known in WO99/54342, WO2000/61739, WO2002/31140, etc., but is not limited thereto.

When the antibody gene is once isolated and introduced into an appropriate host to produce the antibody, a combination of an appropriate host and expression vector can be used.

Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence of the antibody described in this specification and a gene encoding a light chain sequence. When the host cell is transformed, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or they can be inserted into separate expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells, such as COS cells, which are monkey cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 ( ATCC No.CRL-1658) or the dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, L.A. Proc. Natl. Acad. Sci. U.S.A. (1980) 77, p.4126-4220). When using prokaryotic cells, examples include Escherichia coli and Bacillus subtilis. Antibodies can be obtained by introducing the target antibody gene into these cells through transformation, and culturing the transformed cells in vitro. In the above culture methods, the yield may vary depending on the antibody sequence. From among antibodies with equivalent binding activity, the yield can be used as an index to select the one that is easier to produce as a medicine.

The isotype of the antibody used in the present invention is not limited, and examples include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE, but is preferably IgG or IgM, more preferably IgG.

Human IgG1 is one of the five existing human IgG subgroups and has very strong effector functions such as CDC activity through complement fixation and antibody-dependent cell damage activity (Bruggemann et al., J. Exp. Med., 1351-1361 , 1987), and when therapeutic antibodies target molecules that are highly expressed in cancer, they are used as IgG types (trastuzumab, rituximab, etc.), which are It exhibits therapeutic effects by promoting the induction of cell death in cancer cells caused by cell damage through effector functions. Human IgG1 antibodies targeting HLA-DR have been reported to have CDC activity dependent on antibodies possessed by human IgG1, and cynomolgus monkeys died after administration. However, antibody medicine targeting molecules expressed by normal organs has been reported , due to concerns that effective effector function may cause serious side effects (Tawara, T., J. Immunology, 2008, 2294-2298). When IgG1 is used as the isotype of the antibody of the present invention, the IgG1 antibody may have mutations, and the effector function can be adjusted by substituting part of the amino acid residues in the constant region (see WO88/007089, WO94/28027 , WO94/29351). Examples of IgG1 variants that weaken effector functions include IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), and the like.

Human IgG2, one of the five human IgG subgroups that exists, has very weak effector functions such as CDC activity through complement fixation and antibody-dependent cell damage activity (Bruggemann et al., J. Exp. Med., 1351-1361, 1987), one of the IgG types (denosumab) that is used as a therapeutic antibody to target molecules that are expressed in normal organs to avoid toxicity caused by cell damage through effector functions. ), evolocumab, brodalumab).

Among the human IgG subgroups in which there are five types of human IgG4, effector functions such as CDC activity through complement fixation and antibody-dependent cell damaging activity are very weak (Bruggemann et al., J. Exp. Med., 1351-1361, 1987 ), one of the IgG types (OPDIVO®) used as a therapeutic antibody to avoid toxicity caused by cell damage through effector functions when targeting molecules expressed in normal organs. When IgG4 is used as the isotype of the antibody of the present invention, by partially substituting one of the amino acid residues in the constant region, the unique fragmentation of IgG4 is inhibited and the half-life can be extended (see Molecular Immunology, 30, 1 105-108(1993)).

When IgG4 is used as the isotype of the antibody of the present invention, the IgG4 antibody may have mutations. Variants of IgG4 can be listed according to the EU index (Proc Natl Acad Sci US A. 1969, 63 (1), 78-85; Kabat et.al., Sequences of proteins of immunological interest, 1991 Fifth edition) The substitution of phenylalanine at position 234 with alanine (F234A) and the substitution of leucine at position 235 with alanine (L235A) are shown (Parekh et al., mAbs, 310-318, 2012). This variation in antibodies is called FALA variation. IgG4PFALA further weakens the effector function by replacing two essential amino acid residues with alanine on the interaction with FcγRs present in the CH2 functional domain (for example, FcγRI, FcγRII or FcγRIII, etc.).

In addition, IgG4 is unstable due to the formation of SS bonds between antibody heavy chains. In order to improve stability, mutations that promote the formation of SS bonds between antibody heavy chains are introduced. An example of such a mutation is the substitution of serine at position 228 with proline (S228P) shown in the EU index (ANGAL et.al., Molecular Immunology, 105-108, 1993). The mutation of this antibody is called Pro mutation. The above-mentioned FALA mutation and Pro mutation can be introduced simultaneously into the constant region of the antibody of the present invention (Vafa et.al., Methods, 65, 114-126, 2014). The IgG4 heavy chain having FALA mutation is called "IgG4FALA" type heavy chain, the IgG4 heavy chain having Pro mutation is called "IgG4P" type heavy chain, and the IgG4 heavy chain having both FALA mutation and Pro mutation is called It is an "IgG4PFALA" heavy chain.

The constant region of the antibody heavy chain includes CH1, hinge, CH2 and CH3 regions. The CH1 system is defined as EU index 118 to 215, the hinge system is defined as EU index 216 to 230, the CH2 system is defined as EU index 231 to 340, and the CH3 system is defined as EU index 341 to 446. According to the EU index, proline substituted from serine at position 228, alanine substituted from phenylalanine at position 234, and alanine substituted from leucine at position 235 are each equivalent to represent human embedded Proline No. 248, Alanine No. 254, and Alanine No. 255 in the Sequence ID No. 100 of the amino acid sequence of rat_CD147_#84 heavy chain IgG4PFALA represent human chimeric rat_CD147_#101 heavy chain Proline No. 245, Alanine No. 251, and Alanine No. 252 in Sequence ID No. 108 of the amino acid sequence of IgG4PFALA represent the amino acid sequence of human chimeric rat_CD147_#110 heavy chain IgG4PFALA. The sequence identification number 108 includes proline No. 244, alanine No. 250 and alanine No. 251.

Preferable isotypes of the antibody of the present invention include IgG1, IgG2, IgG4, IgG4P, or IgG4PFALA, particularly preferably IgG2, IgG4P, or IgG4PFALA, and still more preferably, IgG2 or IgG4P.

Furthermore, the antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding site of an antibody or a modified product thereof. Fragments of the antibody can be obtained by treating the antibody with proteolytic enzymes such as papain and pepsin, or by changing the antibody gene through genetic engineering and expressing it in appropriately cultured cells. Among such antibody fragments, fragments that retain all or part of the functions of the full-length antibody molecule can be called antigen-binding fragments of the antibody. The functions of antibodies include activation of information transmission related to antigens.

CD147 is also expressed in blood cells including erythrocytes or normal organs necessary for survival (Spring, et al., Eur. J. Immunol., 1997, 891-897), and the antibody utilizes the effector function of the attached antibody. Oncological effects are considered to have a high risk of side effects. It has been reported that red blood cells are actually sensitive to the effector functions (ADCC, CDC, ADCP) generated by antibody binding (Flegel, W., Transfusion, 2015, S47-S58). It is known that anti-erythrocyte antibodies increase in the body and become autoimmune. Autoimmune hemolytic anemia (Gibson, J., Aust. N.Z.J. Med., 1988.625-637). The antibody system of the present invention is a therapeutic antibody targeting CD147, which is also expressed on normal cells, and is characterized by low or almost no ADCC activity, ADCP activity, or CDC activity that may cause severe side effects. detected.

The present inventors discovered for the first time a human CD147 antibody that exhibits anti-tumor activity by activating the cellular signaling of CD147, regardless of the antibody's effector function. The functions retained by the antibodies in the present invention are the binding activity to CD147 and/or the function of activating CD147. The antibody system of the present invention preferably activates related signaling molecules downstream of CD147, for example, FAK, MEK, Erk, JAK/STAT, AKT or MAP kinase (MAPK) or these further downstream related signaling molecules. Activation. The antibody of the present invention preferably activates MAPK or a molecule downstream of MAPK. Preferable MAPK is p38MAPK. As for signaling molecules further downstream of MAPK, for example, HSP27, cxcl8 or SMAD (for example, SMAD2, SMAD3 or SMAD4, preferably SMAD4). "Activation of CD147" includes, for example, an increase in the expression level of p38MAPK mRNA, an increase in the amount of protein expression of p38MAPK, phosphorylation of p38MAPK, and phosphorylation of HSP27 (for example, phosphorylation of Ser82 of HSP27 or HSP27 phosphorylation of Ser15), an increase in cxcl8 mRNA expression, an increase in cxcl8 protein expression, or an increase in rhoBmRNA expression or an increase in rhoB protein expression through SMAD message activation. "Activation of CD147" is preferably an increase in protein expression of p38MAPK, phosphorylation of p38MAPK, phosphorylation of HSP27 (for example, phosphorylation of Ser82 of HSP27 or phosphorylation of Ser15 of HSP27), or expression of cxcl8 mRNA The increase may be due to the increase in the expression of rhoB mRNA activated by SMAD messages.

It is known that SMAD2 or SMAD3 is phosphorylated by the TGFβ receptor when TGFβ binds to the TGFβ receptor (TGFBR1/2), forms a heterotrimer with SMAD4, migrates to the nucleus, and binds to SMAD DNA on the chromosome. The binding sequence (Smad binding element: SBE) binds to the transcriptional regulatory region and positively or negatively controls the mRNA expression of downstream genes (Miyazono, Japanese Medical Journal, 1999, 162-166). Therefore, it is considered that the presence of SMAD2 or SMAD3 is necessary for the activation of SMAD4. SMAD2, SMAD3 and SMAD4 negatively control the expression of TGFb-dependent KLF5 (David et al., Cell, 2016, 164(5), 1015-1030). In SMAD4-deficient pancreatic cancer cells, the inhibitory signals of SMAD2, SMAD3, and SMAD4 through the KLF5 gene are released, and KLF5 protein is expressed. It is known that when SMAD4 is lost and KLF5 is expressed, TGFb-dependent cell death messages (SOX4-dependent) are inhibited (David et al., Cell above).

The inventors found that the human CD147 antibody of the present invention phosphorylates p38MAPK (Fig. 21), phosphorylates HSP27 (Fig. 20), and increases the expression of cxcl8 (Fig. 22(b) and Fig. 23(b)). Therefore, by confirming changes in the gene expression, protein expression, or phosphorylation state of at least one of these molecules before and after administration of the antibody of the present invention, it can be confirmed whether the antibody of the present invention is involved in the activation of CD147.

Furthermore, the present inventors have found that the human CD147 antibody of the present invention exhibits medicinal efficacy against a pancreatic cancer model with protein expression of SMAD4 (Fig. 25); the human CD147 antibody system of the present invention has genetic mutations in SMAD4 and does not express SMAD4 In pancreatic cancer models such as BxPC-3, the anti-tumor effect is partial (~30%) (Fig. 24); and, in tumors after administration of the human CD147 antibody of the present invention, SMAD2, SMAD3 and SMAD4 are downstream molecules (rhoB, Figure 22(c) and Figure 23(c)) were induced. Therefore, whether there is any change in the gene expression or protein expression of rhoB before and after administration of the antibody of the present invention can confirm whether the antibody of the present invention is activated by CD147. In addition, the genome sequence, gene expression or protein expression of SMAD4 in the patient sample is determined using methods well known to those skilled in the art, and patients expressing SMAD4 are selected as target patients for administration of the antibody of the present invention, and can be administered Antibodies of the invention.

In addition, KLF5 expression is known to be high in SMAD4-negative models such as BxPC-3 (David et al., Cell, 2016, 164(5), 1015-1030). The sensitivity of the human CD147 antibody of the present invention in the KLF5-expressing MIA PaCa-2 model ranges from 91% to as low as 20% (Example 26). From this, the present inventors believe that the expression of KLF5 inhibits SMAD2, SMAD3, and SMAD4-dependent cell death messages induced by CD147 antibodies. In liver cancer, ALL, lymphoma, gastrointestinal stromal tumor (GIST), skin cancer, sarcoma, AML or renal cell carcinoma, the expression level of KLF5 is low, and it is expected that the human CD147 antibody of the present invention will be effective in many patients. In addition, the gene expression or protein expression of KLF5 in patient samples can be measured using methods well known to those skilled in the art, and patients with reduced or missing expression of KLF5 are selected as target patients for administration of the antibody of the present invention, and administered Antibodies of the invention. The degree of reduction in KLF5 expression in this case can be determined by methods well known to those of ordinary skill in the art and the conduct of appropriate clinical trials, for example, comparing KLF5 in patients who achieve an effect and in patients who do not. performance, and set appropriate thresholds.

Examples of antibody fragments include Fab, F(ab') ₂ , Fv, single-chain Fv (scFv) and diabodies in which heavy chain and light chain Fv are linked with an appropriate linker. , linear antibodies, and multispecific antibodies formed from antibody fragments, etc. In addition, Fab', which is a monovalent fragment of the variable region of an antibody treated with F(ab') ₂ under reducing conditions, is also included in the fragments of the antibody.

Furthermore, the antibody of the present invention may be a multispecific antibody specific for at least two types of different antigens. Usually such molecules bind to two types of antigens (i.e., bispecific antibodies). The "multispecific antibodies" in the present invention include specificity for more than one (for example, three types) of antigens. of antibodies.

The multispecific antibodies of the present invention may also be antibodies consisting of the entire length or fragments of such antibodies (eg, F(ab') ₂ bispecific antibodies). Bispecific antibodies can also be produced by combining the heavy and light chains (HL pairs) of two types of antibodies. They can also be produced by fusing fusion tumors that produce different monoclonal antibodies to produce bispecific antibody-producing fusion cells. (Millstein et al., Nature (1983) 305, p. 537-539).

The antibody of the present invention may also be a single-chain antibody (also described as scFv). Single-chain antibodies can be obtained by connecting the heavy chain variable region and the light chain variable region of the antibody with a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore eds.; Springer Verlag, New York, p.269-315 (1994); Nature Biotechnology (2005), 23, p.1126-1136). In addition, a BiscFv fragment produced by binding two scFvs with a polypeptide linker can also be used as a bispecific antibody.

Methods for producing single-chain antibodies are well known in the art (for example, refer to U.S. Patent No. 4,946,778, U.S. Patent No. 5,260,203, U.S. Patent No. 5,091,513, U.S. Patent No. 5,455,030, etc.). In this scFv, the heavy chain variable region and the light chain variable region are connected through a linker that does not form conjugation, preferably through a polypeptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, p. 5879-5883). The heavy chain variable region and light chain variable region in scFv can come from the same antibody or from different antibodies. As a polypeptide linker connecting the variable regions, for example, any single-chain peptide consisting of 12 to 19 residues can be used.

The DNA encoding scFv can be obtained by combining the DNA encoding the heavy chain or heavy chain variable region of the aforementioned antibody and the DNA encoding the light chain or light chain variable region, and combining the sequences encoding them. All or the DNA portion of the desired amino acid sequence is used as a template, and primer pairs defining the two ends are used to amplify by PCR. Then, the DNA encoding the polypeptide linker portion and its two ends are combined with the heavy chain and the heavy chain respectively. The combination of primer pairs specified in the way of light chain connection is obtained by increasing the amplitude.

Furthermore, once DNA encoding scFv is produced, an expression vector containing the expression vector and a host transformed from the expression vector can be obtained according to ordinary methods. Furthermore, by using this host, scFv can be obtained according to ordinary methods. These antibody fragments can be obtained and expressed in the same manner as described above, and can be produced by a host.

The antibody of the present invention can be quantified multiple times to increase the affinity for the antigen. The multiplexed antibody may be a single type of antibody or a plurality of antibodies that recognize plural epitopes of the same antigen. Examples of methods for multiplexing antibodies include binding of the IgG CH3 domain to two seFvs, binding to streptavidin, introduction of a helix-turn-helix motif, etc. .

The antibody of the present invention can be a mixture of multiple anti-CD147 antibodies with different amino acid sequences, that is, a polyclonal antibody. An example of a polyclonal antibody is a mixture of multiple types of antibodies with different CDRs. As such polyclonal antibodies, a mixture of cells producing different antibodies can be cultured, and antibodies purified from the culture can be used (see WO2004/061104).

The antibody of the present invention may be an antibody having 80% to 99% identity (or identity) with the heavy chain and/or light chain of the above-mentioned antibody. By combining sequences showing high identity with the above-mentioned heavy chain amino acid sequence and light chain amino acid sequence, it is possible to select an antibody that has an antigen-binding ability equal to that of each of the above-mentioned antibodies and activates CD147, preferably MAPK. Antibodies that activate and activate downstream signaling molecules of MAPK. Generally speaking, the identity is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining the amino acid sequence of the heavy chain and/or light chain with one to several amino acid residues substituted, deleted and/or added, the amino acid sequence having the above-mentioned characteristics can be selected. Each antibody has the same various effects. The number of substituted, deleted and/or added amino acid residues is generally less than 10 amino acid residues, preferably less than 5 to 6 amino acid residues, more preferably 2 to 3 amines amino acid residue or less, preferably 1 amino acid residue.

Furthermore, it is known that the lysine residue at the carboxyl terminus of the heavy chain of antibodies produced by mammalian cultured cells is deleted (Tsubaki et.al., Int. J. Biol. Macromol, 139-147, 2013). However, these deletions and modifications of heavy chain sequences have no impact on the antigen-binding ability and effector functions (activation of complement or antibody-dependent cellular damage, etc.) of the antibody. Therefore, the present invention also includes antibodies that accept this modification, deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and such deletions that are amidated (e.g., proline residues at the carboxyl terminus) Aminated heavy chain), etc. However, as long as it retains the antigen-binding ability and the function of activating downstream related signaling molecules of CD147, the deletion of the carboxyl terminus of the heavy chain of the antibody related to the present invention is not limited to the above-mentioned types. The double-stranded heavy chain constituting the antibody related to the present invention may be any one selected from the group consisting of the full length and the above-mentioned deletion, or may be a combination of any two. The quantitative ratio of each deletion may be affected by the type of mammalian cultured cells that produce the antibody related to the present invention and the culture conditions. However, as the main component of the antibody related to the present invention, double-stranded heavy chains can be cited. On both sides, one amino acid residue at the carboxyl terminus is missing.

The identity between the two types of amino acid sequences can be determined by using the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can also be used by accessing it on the Internet at www.ncbi.nlm.nih.gov/blast. Furthermore, according to the above Blast algorithm, the percentage values of the two types of identity (Identity (or Identities)) and positivity (Positivity (or Positivities)) are calculated. The former is the value when the amino acid residues are consistent between the two types of amino acid sequences for which identity should be determined, and the latter is the value when similar amino acid residues also take into account the chemical structure. In this specification, the value of Identity (identity) when the amino acid residues are identical is defined as the value of identity.

As modified antibodies, antibodies bound to various molecules such as polyethylene glycol (PEG) can also be used.

The antibodies of the present invention may further be those in which these antibodies form conjugates (Immunoconjugate) with other pharmaceutical agents. Examples of such antibodies include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological effect (Nature Biotechnology (2005) 23, p. 1137-1146).

The antibodies obtained can be purified to homogeneity. For isolation and purification of antibodies, the isolation and purification methods commonly used for proteins may be used. For example, by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric point electrophoresis, etc., antibodies can be separated and purified (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but It is not limited to this.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography, and the like.

Such chromatography can be performed using liquid chromatography such as HPLC, FPLC, and the like.

Examples of columns used for affinity chromatography include protein A (Protein A) columns and protein G (Protein G) columns.

For example, examples of columns using protein A columns include Hyper D, POROS, Sepharose F.F. (GE HEALTHCARE), and the like.

Antibodies can also be purified using a carrier that immobilizes the antigen and takes advantage of its binding ability to the antigen.

(Medicines containing anti-CD147 antibodies)

The anti-CD147 antibody of the present invention can be obtained from the anti-CD147 antibody obtained by the method described in the above item "Preparation of anti-CD147 antibody". The antibodies thus obtained can be used as tumor and/or cancer therapeutic and/or preventive agents.

The anti-CD147 antibody of the present invention has excellent anti-tumor activity and is useful as a tumor or cancer therapeutic drug. The anti-CD147 antibody of the present invention exhibits excellent anti-tumor effects even against gemcitabine-resistant cancer cells or sorafenib-low-susceptibility cancer cells. The anti-CD147 antibody of the present invention targets chronic myelogenous leukemia cells and exhibits significantly stronger efficacy than imatinib.

Tumors that can be treated by the anti-CD147 antibody of the present invention or medicines containing the antibody are not particularly limited as long as they express CD147, but preferred examples include pancreatic cancer, liver cancer, gastric cancer, and colorectal cancer. Kidney cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, thyroid cancer, skin cancer, head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumor, gastrointestinal stromal tumor (GIST), leukemia (e.g., acute myeloid Leukemia (AML), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL) or acute lymphoblastic leukemia (ALL)), lymphoma, or malignant lymphoma (e.g., B-cell lymphoma, non-Hodgkin's Lymphoma or diffuse large B-cell lymphoma (DLBCL)), preferably pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, renal cancer, leukemia, acute myeloid leukemia (AML) ), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell B-cell Lymphoma (DLBCL).

Furthermore, examples of tumors that can be treated by the antibodies of the present invention or medicines containing the antibodies include tumors that express SMAD-positive CD147. Examples of tumors that express SMAD-positive CD147 include, for example, tumors that express SMAD-positive CD147. CD147 liver cancer or pancreatic cancer. The anti-CD147 antibody of the present invention or the pharmaceutical system containing the antibody is preferably administered to patients with confirmed expression of CD147 and/or SMAD. As for SMAD, SMAD2, SMAD3 and/or SMAD4 are preferred, and SMAD4 is more preferred. Preferably, the performance of at least one of SMAD2 or SMAD3 is confirmed based on the confirmation of the performance of SMAD4.

Alternatively, tumors that can be treated by the antibody of the present invention or a medicine containing the antibody include tumors in which the expression of KLF5 is reduced or deleted, and tumors in which the expression of KLF5 is reduced or deleted include liver cancer and ALL. , lymphoma, gastrointestinal stromal tumor (GIST), skin cancer, sarcoma, AML or renal cell carcinoma. The anti-CD147 antibody of the present invention or a medicine containing the antibody is preferably administered to patients who are confirmed to have reduced or deleted expression of KLF5.

The anti-CD147 antibody of the present invention can also be administered with 2, 3 or more other therapeutic agents depending on the purpose of treatment, and can be administered simultaneously by encapsulating the other therapeutic agents in the same preparation. Other therapeutic agents and anti-CD147 antibodies can be administered simultaneously by encapsulating them in the same formulation. Alternatively, anti-CD147 antibodies and other therapeutic agents may be enclosed in separate preparations and administered simultaneously. Furthermore, other drugs can also be administered separately before and after the anti-CD147 antibody. That is, a therapeutic agent containing an anti-CD147 antibody or an antigen-binding fragment of the antibody as an active ingredient may be administered after other therapeutic agents are administered, or a therapeutic agent containing an anti-CD147 antibody or the antibody as an active ingredient may be administered. After the therapeutic agent of the antigen-binding fragment is administered, the other therapeutic agent is administered.

The present invention also provides pharmaceutical compositions containing a therapeutically and/or preventively effective amount of anti-CD147 antibodies and pharmaceutically acceptable diluents, carriers, co-solvents, emulsifiers, preservatives and/or auxiliaries.

The present invention also provides an anti-tumor therapeutic agent containing a therapeutically and/or preventively effective amount of anti-CD147 antibody and a therapeutically and/or preventively effective amount of at least one, and a pharmaceutically acceptable diluent, carrier, co-solvent, and emulsifier. , preservatives and/or auxiliary pharmaceutical compositions.

The substance used in the acceptable preparation of the pharmaceutical composition of the present invention is preferably one that is non-toxic to the person to whom the pharmaceutical composition is administered in terms of dosage or concentration.

The pharmaceutical composition of the present invention may contain preparations for changing or maintaining pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, sustained release rate, absorption rate, and penetration rate. material. Examples of substances used in preparations include, but are not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, arginine or lysine, and antibacterial agents , antioxidants such as ascorbic acid, sodium sulfate or sodium bisulfite, buffers such as phosphoric acid, citric acid, boric acid buffer, sodium bicarbonate, Tris-hydrochloric acid (Tris-Hcl) solution, mannitol or glycine, etc. Fillers, chelating agents such as ethylenediaminetetraacetic acid (EDTA), caffeine, polyvinylpyrrolidine, β-cyclodextrin or hydroxypropyl-β-cyclodextrin, etc., glucose, mannose or Extenders such as dextrin, other carbohydrates such as monosaccharides and disaccharides, colorants, flavors, diluents, emulsifiers or hydrophilic polymers such as polyvinylpyrrolidine, low molecular weight polypeptides, salts forming counter ions, Preservatives such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide , solvents such as glycerol, propylene glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspending agents, polysorbate esters such as sorbitan ester, polysorbate 20 or polysorbate 80 , triton, trihydroxyaminomethane (tromethamine), surfactants such as lecithin or cholesterol, stabilizing enhancers such as sucrose or sorbitol, sodium chloride, potassium chloride, mannitol and sorbitol Elasticity enhancers, delivery agents, excipients, and/or pharmaceutical auxiliaries such as sugar alcohols. The added amount of these preparation substances is 0.01 to 100 times the weight of the anti-CD147 antibody, especially 0.1 to 10 times is preferred. The composition of the suitable pharmaceutical composition in the preparation can be appropriately determined by those with common knowledge in the technical field according to the applicable disease, applicable route of administration, etc.

Excipients or carriers in pharmaceutical compositions can be liquid or solid. Suitable excipients or carriers may be water for injection or physiological saline, artificial cerebrospinal fluid, or other substances commonly used for parenteral administration. Neutral physiological saline or physiological saline containing serum albumin can also be used as the carrier. The pharmaceutical composition may contain Tris buffer at pH 7.0-8.5, acetic acid buffer at pH 4.0-5.5, and citrate buffer at pH 3.0-6.2. In addition, these buffer solutions may also contain sorbitol or other compounds. Examples of the pharmaceutical composition of the present invention include pharmaceutical compositions containing anti-CD147 antibodies, and pharmaceutical compositions containing anti-CD147 antibodies and at least one anti-tumor therapeutic agent. The pharmaceutical compositions of the present invention are those having a selected composition and A pharmaceutical product of the necessary purity is prepared as a freeze-dried product or liquid. Pharmaceutical compositions containing anti-CD147 antibodies and pharmaceutical compositions containing anti-CD147 antibodies and at least one anti-cancer agent therapeutic agent can also be shaped into freeze-dried products using appropriate excipients such as sucrose.

The pharmaceutical composition of the present invention can be prepared for parenteral administration or for oral absorption through the digestive tract. The composition and concentration of the preparation can be determined according to the administration method. When the antibody of the present invention is administered to humans, approximately 0.1 to 100 mg/kg may be administered once or multiple times within 1 to 180 days. However, the dosage and frequency of administration are generally determined by taking into consideration the gender, weight, age, symptoms, severity, side effects, etc. of the patient, and are not limited to the above dosage and usage.

Examples of the forms of the pharmaceutical composition of the present invention include intravenous injections, suppositories, nasal preparations, sublingual preparations, and transdermal absorption preparations. The route of administration may be an oral route or a parenteral route. Examples of the non-oral route include intravenous, intraarterial, intramuscular, intrarectal, intramucosal, and intradermal routes.

The antibodies of the present invention or antigen-binding fragments of the antibodies, drug complexes containing the same, bispecific antibodies containing the same, or pharmaceutical compositions containing the same can be used to select patients to be administered the same A combination of biomarkers is provided. Such antibodies or pharmaceutical compositions can be combined with means for detecting biomarkers and provided as a set, and the provision of antibodies or pharmaceutical compositions and the provision of biomarkers can also be performed separately. By using biomarkers, the antibodies or pharmaceutical compositions of the present invention can be administered to patient groups in which higher efficacy of the antibodies of the present invention can be expected.

The present invention relates to a method for predicting response to cancer treatment, which includes using a biological sample derived from a cancer patient, measuring the expression of SMAD4 or the expression of KLF5 contained in the biological sample, and detecting Patients with SMAD4 or patients whose expression of KLF5 is reduced or deleted are determined to be responsive to cancer treatment using the antibody of the present invention or a functional fragment of the antibody or the pharmaceutical composition of the present invention; a method for screening cancer treatment subjects A method comprising using a biological sample derived from a cancer patient, detecting the expression of SMAD4 or the expression of KLF5 in the biological sample, and screening patients with detected SMAD4 or reduced or missing expression of KLF5. Those who are candidates for cancer treatment using the antibody of the present invention or a functional fragment of the antibody or the pharmaceutical composition of the present invention; a method of cancer treatment that includes using a biological sample derived from a cancer patient to detect the biological The expression of SMAD4 or the expression of KLF5 contained in the test sample is administered to patients whose SMAD4 is detected or to patients whose expression of KLF5 is reduced or missing, and the antibody of the present invention or a functional fragment of the antibody or the medicine of the present invention is administered composition; or, a kit for determining the response to cancer treatment using the antibody of the present invention or a functional fragment of the antibody or the pharmaceutical composition of the present invention, which at least includes a detection source Means of expression of SMAD4 or expression of KLF5 in biological samples of cancer patients.

In this specification, "biological samples" refer to tissues, liquids, cells, and mixtures thereof isolated from an individual. Examples include tumor biopsy, bone marrow fluid, intrapleural fluid, intraperitoneal fluid, lymph fluid, Skin slices, blood, urine, feces, sputum, respiratory organs, intestines, urogenital tract, saliva, milk, digestive organs, and cells collected therefrom, but are not limited to these. The "biological sample" preferably exemplifies a sample containing cancer cells, and more preferably exemplifies tissue or cells obtained from resection or biopsy, or cells derived from intrapleural fluid or intraperitoneal fluid. A further preferred biological sample is a sample containing cancer cells or cancer tissue.

"Expression of SMAD4" can be detected or measured using methods well-known to those with ordinary knowledge in the art to perform genome sequencing, gene expression or protein expression of SMAD4, for example, RNA sequencing, microarray, Genome sequencing, immune analysis.

The detection or determination of "expression of KLF5" can use methods well known to those with ordinary knowledge in the technical field to detect the genome sequence, gene expression or protein expression of KLF5, for example, IHC, RNA sequencing, microarray , genome sequencing, and immune analysis. "Reduced expression of KLF5" refers to a lower expression level compared to a control (eg, expression level in healthy subjects or non-cancerous tissue from the same patient). Alternatively, the degree of reduction in KLF5 expression that can be determined to be responsive to cancer treatment using the antibody or pharmaceutical composition of the present invention can be determined by methods well known to those of ordinary skill in the art and the conduct of appropriate clinical trials. For example, the expression amount of KLF5 in patients who have obtained an effect and in patients who have not obtained an effect is compared, and an appropriate threshold is set. Thus, "reduced performance of KLF5" means, for example, lower than the threshold so set.

[Example]

The present invention will be specifically described below through examples, but the present invention is not limited thereto. Furthermore, these are not limited in any sense. In addition, in the following examples, unless otherwise specified, each operation related to gene manipulation is based on "Molecular Cloning" (Sambrook, J., Fritsch, E.F. and Maniatis, T., Cold Spring Harbor Laboratory Press published in 1989) and other methods recorded in experimental books used by those with ordinary knowledge in the technical field, or in the case of using commercially available reagents or kits, follow the commercially available reagents or kits. Follow the product instructions. In addition, in this specification, reagents, solvents and starting materials not specifically described can be easily obtained from commercially available sources. In this example, the human pancreatic cancer cell line MIA PaCa-2 uses ATCC Cat. CRL-1420, and the human pancreatic cancer cell line PANC-1 uses ATCC Cat. CRL-1469.

(Example 1) Preparation of mouse and rat antibodies using cellular immunity 1)-1 Production of CD147 expression vector

Gateway LR Clonase was used to react the commercially available human CD147 gene (BSG variant2/CD147v2) clone strain IOH3378 (Invitrogen) with the mammalian cell expression vector pcDNA-DEST40 (Invitrogen) to produce the human CD147v2 expression vector (pcDNA-DEST40 -CD147v2).

The commercially available mammalian cell expression vector pCMV6-XL5-hBSGv1 (Origene Company, Cat. SC303059) of the human CD147 gene (BSG variant1/CD147v1) was purchased as the human CD147v1 expression vector.

As a cynomolgus monkey CD147 expression vector, pCMV3-cynoBSG (Sino Biological Inc., Cat. CG90636-UT) was purchased.

As a mouse CD147v2 expression vector, pCMV3-mBSGv2 (Sino Biological Inc., Cat. MG50332-UT) was purchased.

1)-2 Preparation of mouse fusion tumors

BALB/cAnNCrlCrlj mice (Charles River Corporation, Japan) aged 4 to 6 weeks were used. On days 0, 7, 15 and 24, 5×10 ⁶ LNCaP cells (ATCC, CRL-1740) exfoliated by Versene (Thermo Fisher Scientific) were suspended in PBS and administered to the back. Subcutaneous. On the 31st day, 5×10 ⁶ of the same cells were intravenously administered, and spleen cells were harvested and used for fusion tumor production on the same day. Spleen cells and mouse myeloma P3X63Ag8U.1 cells (ATCC, CRL-1597) were fused using PEG4000 (IBL Company) to prepare fusion tumors. For the isolation and culture of fusion tumors, ClonaCell-HY MediumD (STEMCELL TECHNOLOGIES) and ClonaCell-HY MediumE (STEMCELL TECHNOLOGIES) were used.

1)-3 Preparation of rat fusion tumors

Use 7-week-old WKY/Izm (Japan SLC Co., Ltd.). 1×10 ⁷ human pancreatic cancer cell line PANC-1 was immunized in the buttocks and intestinal bone lymph node cells were harvested 13 days later for fusion tumor production. Using the LF301 cell fusion device (BEX CO., LTD.), rat spleen cells and mouse myeloma SP2/0-Ag14 cells (ATCC, CRL-1581) were fused to produce fusion tumors. For the isolation and culture of fusion tumors, ClonaCell-HY MediumD (STEMCELL TECHNOLOGIES) and ClonaCell-HY MediumE (STEMCELL TECHNOLOGIES) were used.

1)-4 Antigen identification using ELISA

Human CD147Fc fusion protein (Sino Biological, Cat. 10186-H02H) and mouse CD147Fc fusion protein (Sino Biological, Cat. 50332-M03H) were used. Human CD147Fc fusion protein and mouse CD147Fc fusion protein were added to PBS buffer, dissolved on ice, and adjusted to 1 μg/ml. Add 100 μl of the same protein lysate to a 96-well plate (NUNC, Cat. 442404), store it at 4°C overnight, and coat the wells with CD147Fc fusion protein. The protein lysate was removed, and the wells were blocked with PBS buffer containing 1% BSA (Research Organics, Cat. 1334A) for 2 hours at 4°C. After washing the well three times with PBS buffer containing 0.05% Tween20 (ATTO company, Cat. WSE-7235), the fusion tumor culture supernatant prepared in Examples 1)-2 and 1)-3 was washed with PBS. Buffer was diluted 20-fold, added to each well, and heated at room temperature for 1 hour. After washing the well three times with PBS buffer containing 0.05% Tween20 (ATTO, Cat. WSE-7235), add 100 μl of anti-rat-Fab2-igG- diluted 50,000 times with PBS buffer containing 1% BSA. HRP (Jackson ImmunoResearch Company, Cat. 112-036-072) for 30 minutes, shaking at room temperature. After washing the well 5 times with PBS buffer containing 0.05% Tween20 (ATTO Company, Cat. WSE-7235), add 100 μl of HRP enzyme chromogenic reagent (eBioscience Company, Super AquaBlue ELISA substrate, Cat. 00-4203 ), raise the temperature to room temperature for 10 to 20 minutes, and measure the absorbance at 405 nm with a flat plate reader (Envision, PerkinElmer Company). Calculate the average value of the measured absorbance values of 2 to 3 wells. Antibodies with an absorbance of more than 2 times the measured value in the control wells without primary antibodies are judged to have binding (+), and those with an absorbance lower than 2 times are judged to have binding (+). It was judged as no binding (-) and summarized in Table 1. In the culture supernatants of LN22R8, 2P1A6, 2P3A9, 2P3G8, 2P8C12, 2P10F2, 2P2D7, 2P2D10, and 2P1B7, specific color development of the wells coated with human CD147Fc fusion protein was confirmed. In LN24R7, 2P5F5, 2P6A2, and 2P3G8, specific color development was confirmed in the coated wells encoding human and mouse CD147Fc fusion proteins.

1)-5 Preparation of monoclonal antibodies and determination of antibody isotypes

For the fusion tumors that were confirmed to produce anti-human CD147 antibodies in Examples 1)-4 and were stable for culture, a commercially available isotyping kit was used to determine the isotype of the antibodies contained in the culture supernatant and are shown in Table 2. These fusion tumors were cultured using a CL-1000 flask (Becton Dickinson Co., Ltd., Japan), and a fusion tumor culture supernatant containing a monoclonal antibody was prepared.

1) Purification of -6 monoclonal antibodies

The antibody was purified from the culture supernatant prepared in Example 1)-5. For the anti-human CD147 mouse monoclonal antibody, purification was performed in a 1-stage step using rProtein A affinity chromatography (at 4~6°C). The buffer replacement step after rProtein A affinity chromatography purification is performed at 4~6°C. First, the culture supernatant was applied to a column packed with MabSelectSuRe (manufactured by GE Healthcare Bioscience) equilibrated with PBS. After all the culture medium is placed in the column, wash the column with PBS at least twice the capacity of the column. Next, the solution was eluted with 2M spermine hydrochloride solution (pH 4.0), and the fraction containing the antibody was collected. This fraction was subjected to solution replacement of HBSor (25mM Histidine/5% Sorbitol/pH6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). Concentrate with Centrifugal UF Filter Device VIVASPIN20 (fractionated molecular weight UF10K, Sartorius, 4°C) to adjust the IgG concentration to 4.9 mg/ml. Finally, it was filtered with Minisart-Plus filter (Sartorius Company) and used as a purified sample.

For the anti-human CD147 rat monoclonal antibody, purification was performed in one stage using Protein G affinity chromatography (at 4~6°C). The buffer replacement step after Protein G affinity chromatography purification is performed at 4~6°C. First, the culture supernatant of the fusion tumor was applied to a column filled with ProteinG (GE Healthcare Bioscience) equilibrated with PBS. After all the culture supernatant is placed in the column, wash the column with PBS at least twice the capacity of the column. Next, the solution was eluted with a 0.1M glycine/hydrochloric acid aqueous solution (pH 2.7), and the antibody-containing fractions were collected. 1M Tris-HCl (pH9.0) was added to the collected fractions to adjust the pH to 7.0~7.5, and then HBSor ( 25mM Histidine/5% Sorbitol/pH6.0) buffer was substituted and concentrated at the same time to adjust the antibody concentration to 1 mg/mL or more. Finally, it was filtered with Minisart-Plus filter (Sartorius Company) and used as a purified sample.

1)-7 Antibody screening by in vivo anti-tumor activity assay

1×10 ⁷ human pancreas strain PANC-1 was suspended in PBS and transplanted subcutaneously into the axilla of NOD-scid mice (Japanese Charles River Company, NOD.CB17-Prkdc<scid>/J). Based on tumor volume classification, mouse anti-CD147 antibody (LN22R8) and rat anti-CD147 antibody (2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2) were administered to the load at 10 mg/kg 27, 34, and 41 days after transplantation. intraperitoneal cavity of cancerous mice (n=6). 27 and 34 days after transplantation, rat anti-CD147 antibody (2P2D6) was administered into the abdominal cavity of cancer-bearing mice at 10 mg/kg (n=6). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 1(a)~(c). In the figure, the changes in tumor volume are combined with the mean and standard error. The results of 2P2D6 antibody, 2P3G8 antibody and 2P2D10 antibody are shown in Figure 1(a). The tumor growth inhibition rates 48 days after transplantation on the final measurement day were 10%, 45%, and 40% for the 10 mg/kg group. The results of LN22R8 antibody, 2P1A6 antibody and 2P1B7 antibody are shown in Figure 1(b). The tumor growth inhibition rates 48 days after transplantation on the final measurement day were 50%, 26%, and 24% in the 10 mg/kg group. The results of 2P8C12 antibody and 2P10F2 antibody are shown in Figure 1(c). The tumor growth inhibition rates 48 days after transplantation on the final measurement day were -2% and 62% in the 10 mg/kg group.

1)-8 Analysis of cross-species species of CD147 antibodies

Into CHO-K1 cells (ATCC, CCL-61), Lipofectamine 2000 (Thermofishers scientific, Cat. 11668-019) was used to introduce pcDNA-DEST40-CD147v2 or pCMV3-cynoBSG prepared in Example 1)-1, 1 In the future, mouse anti-human CD147 antibody (LN22R8), or rat anti-human CD147 antibody (2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2, 2P2D6), treated at 10 μg/ml, using anti-mouse IgG-FITC (MP Biomedical, Cat. 554936) or anti-rat IgG-PE (BD Biosciences, Cat. 550767), can fluorescently detect the binding of each antibody to CD147-expressing CHO-K1 cells. The expression of human and cynomolgus CD147 in CHO-K1 cells can be detected by fluorescence through the combination of commercially available anti-CD147 antibodies (MEM-M6/1, Ab serotec, Cat.MCA28822). The above cells were measured with a flow cytometer (CantoII, BD Bioscience), and the results are summarized in Figures 2-1 to 3. The vertical axis of the figure represents the number of cells, and the horizontal axis represents the intensity of the fluorescent signal.

Commercially available anti-CD147 antibodies (MEM-M6/1), mouse anti-human CD147 antibodies (LN22R8), rat anti-CD147 antibodies (2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2, 2P2D6), any of which All CD147 antibodies show binding to human CD147-expressing CHO-K1 cells (Figures 2-1~3).

Although the commercially available anti-CD147 antibody (MEM-M6/1) shows binding to cynomolgus monkey CD147-expressing CHO-K1 cells, mouse anti-human CD147 antibody (LN22R8), rat anti-human CD147 antibody (2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2, 2P2D6), none of the CD147 antibodies showed binding to cynomolgus monkey CD147 CHO-K1 cells (Figures 2-1~3).

Furthermore, none of the anti-CD147 antibodies showed binding to mouse CD147-expressing CHO-K1 cells (data not documented).

1)-9 epitope analysis Preparation of human CD147 allogeneic expression vector for epitope analysis

According to the results of BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi), the amino acid sequences of CD147 in cynomolgus monkeys and humans are 81% identical. It is assumed that limited amino acid differences have an impact on CD147 Antibody binding has a direct impact on the identification of epitopes, and the hypothesis of anti-tumor epitopes was made using some variants grafted on amine sequences that differ between species. Taking the amino acid sequence shared by hCD147v1 and v2 as a target, sequence comparison of cynomolgus monkey and human CD147 was performed, and the amino acid regions were classified into 9 regions with different amino acid regions between species, designated as mu1 to mu9 (Figure 3). The cDNA sequence of human CD147 variant 2, which was artificially synthesized in the expression vector of CD147 variant and confirmed on the cell membrane, was used to introduce the FLAG sequence at the N terminus, and was introduced into the plasmid of the pcDNA3.1 vector to produce Signal-N-Flag-hCD147v2_pcDNA3. 1 (produced by Genscript). Then, using the human CD147 gene in the same plasmid, targeting the CD147 amino acid sequences mu1~mu9 of cynomolgus monkeys, nine human-cynomolgus chimeric CD147 expression vectors were introduced as amino acid substitution mutations through DNA substitution. , to produce hCD147-mu1_pcDNA3.1, hCD147-mu2_pcDNA3.1, hCD147-mu3_pcDNA3.1, hCD147-mu4_pcDNA3.1, hCD147-mu5_pcDNA3.1, hCD147-mu6_pcDNA3.1, hCD147-mu7_pcDNA3.1, hCD147-mu8_pcDNA3.1 , hCD147-mu9_pcDNA3.1 (produced by Genscript Company).

1)-10 Specification of anti-tumor epitope regions using variants

Human CD147, cynomolgus monkey CD147 or human-cynomolgus chimeric CD147 expression vectors were introduced into CHO-K1 cells (ATCC, CCL- 61), 1 day later, anti-human CD147 mouse antibody (LN22R8), rat anti-human CD147 antibody (2P1A6, 2P1B7, 2P3G8, 2P2D10, 2P8C12, 2P10F2, 2P2D6) were treated at 10 μg/ml, and anti-mouse IgG-PE was used (DAKO, Cat. R480), anti-rat IgG-PE (BD, #550767), to investigate the binding of anti-CD147 antibodies to CD147-expressing CHO-K1 cells. The expression of CD147 protein was confirmed using a commercially available anti-FLAG antibody (anti-Flag M2, SIGMA Company, Cat. F4049-.2MG). Measurement was performed with a flow cytometer (CantoII, BD Bioscience), and the results were summarized in Table 3. Samples in which a 10-fold increase in fluorescence signal was observed compared to control cells not treated with the primary antibody were judged to be binding positive (+). For samples in which an increase in fluorescence signal less than 10 times was observed, the binding was judged to be weakly positive (±). Compared with the control cells that were not treated with the primary antibody, samples in which no increase in fluorescence signal was observed were judged to be binding negative (-).

In Examples 1)-7, any of the antibody systems of antibodies 2P3G8, 2P10F2, 2P2D10, and LN22R8, which had an anti-tumor effect of more than 40%, lost binding to CD147 having a mu3 mutation. It is suggested that the important epitope in the anti-tumor effect is the m3 region.

1)-11 Determination of nucleotide sequences of cDNA encoding variable regions of LN22R8 and 2P10F2 antibodies 1)-11-1 Determination of the nucleotide sequence of the cDNA encoding the variable region of the LN22R8 antibody 1)-11-1-1 Preparation of total RNA from LN22R8 antibody-producing fusion tumors

In order to amplify the cDNA encoding the variable region of the LN22R8 antibody, fusion tumors were generated from the LN22R8 antibody, and total RNA was prepared using TRIzol Reagent (Ambion).

1)-11-1-2 Amplification and sequence determination of cDNA encoding the light chain variable region of the LN22R8 antibody using 5’-RACE PCR

Amplification of the cDNA encoding the light chain variable region was carried out using approximately 1 μg of the total RNA prepared in Example 1)-11-1-1 and SMARTer RACE 5'/3' Kit (Clontech). As primers for PCR amplification of cDNA encoding the variable region of the light chain gene of the LN22R8 antibody, UPM (Universal Primer A Mix: attached to the SMARTer RACE 5'/3' set) and a well-known mouse light chain were used An introduction designed by the sequence of constant regions.

The cDNA encoding the variable region of the light chain amplified by 5'-RACE PCR was selected and cloned into plastids, and then sequence analysis of the nucleotide sequence of the cDNA encoding the variable region of the light chain was performed.

The determined nucleotide sequence of the cDNA encoding the variable region of the light chain of the LN22R8 antibody is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. CDRL1, CDRL2 and CDRL3 of the light chain variable region of the LN22R8 antibody are shown in Sequence Identification Numbers 11, 12 and 13, respectively.

1)-11-1-3 Amplification and sequence determination of cDNA encoding the heavy chain variable region of LN22R8 antibody by 5’-RACE PCR

Amplification of the cDNA encoding the heavy chain variable region was carried out using approximately 1 μg of the total RNA prepared in Example 1)-11-1-1 and SMARTer RACE 5'/3' Kit (Clontech). As primers for PCR amplification of the cDNA variable region encoding the heavy chain gene of the LN22R8 antibody, UPM (Universal Primer A Mix: attached to the SMARTer RACE 5'/3' set) and a well-known mouse heavy chain were used An introduction designed by the sequence of constant regions.

The cDNA encoding the variable region of the heavy chain amplified by 5'-RACE PCR was selected and cloned into plastids, and then sequence analysis of the nucleotide sequence of the cDNA encoding the variable region of the heavy chain was performed.

The determined nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the LN22R8 antibody is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the LN22R8 antibody are shown in Sequence ID Nos. 14, 15 and 16, respectively.

1)-11-2 Determination of the nucleotide sequence of the cDNA encoding the variable region of the 2P10F2 antibody

Implemented in the same manner as in Example 1)-11-1. However, as a primer for PCR amplification of the cDNA encoding the variable region of the light chain gene, UPM (Universal Primer A Mix: attached to the SMARTer RACE 5'/3' set) and the well-known constant constant of the rat light chain were used. The primers designed based on the sequence of the region were used as primers for PCR amplification of cDNA encoding the variable region of the heavy chain gene, using UPM (Universal Primer A Mix: attached to the SMARTer RACE 5'/3' set), and well-known Primers designed from the sequence of the constant region of the rat heavy chain.

The determined nucleotide sequence of the cDNA encoding the variable region of the light chain of the 2P10F2 antibody is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18. CDRL1, CDRL2 and CDRL3 of the light chain variable region of the 2P10F2 antibody are shown in Sequence ID Nos. 21, 22 and 23, respectively. The nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20. CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the 2P10F2 antibody are shown in Sequence ID Nos. 24, 25 and 26, respectively.

1)-12 Production of LN22R8 human chimeric antibody expression vector 1)-12-1 Construction of human chimeric and humanized light chain expression vector pCMA-LK

Using the In-Fusion HD PCR cloning kit (Clontech Company), plasmid pcDNA3.3-TOPO/LacZ (Invitrogen Company) was digested with restriction enzymes XbaI and PmeI to obtain a fragment of about 5.4 kb, and included the sequence identification number The DNA fragments encoding the human light chain message sequence and the DNA sequence of the human kappa chain constant region shown in 27 were combined to create pcDNA3.3/LK.

pCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3/LK.

1)-12-2 Construction of human chimeric and humanized IgG1 heavy chain expression vector pCMA-G1

Using the In-Fusion HD PCR cloning kit (Clontech), pCMA-LK was digested with XbaI and PmeI to remove the light chain message sequence and the DNA fragment of the human kappa chain constant region, as well as the code shown in Sequence ID No. 28 DNA fragments of the human heavy chain message sequence and the DNA sequence of the amino acid sequence of the human IgG1 constant region were combined to construct pCMA-G1.

1)-12-3 Construction of human chimeric and humanized IgG2 heavy chain expression vector pCMA-G2

Using the DNA fragment encoding the human heavy chain message sequence and the amino acid sequence of the human IgG2 constant region shown in Sequence ID No. 29, pCMA-G2 was constructed in the same manner as in Example 1)-12-2.

1)-12-4 Construction of human chimeric LN22R8 light chain expression vector

The cDNA encoding the variable region of the light chain of LN22R8 obtained in Example 1)-11-1-2 was used as a template to use the designed primer in In-fusion selection. By performing PCR, the cDNA encoding the light chain was DNA fragments of the cDNA in the variable region of the chain are amplified. The human chimeric LN22R8 light chain expression vector was constructed by inserting the amplified DNA fragment into the place where pCMA-LK was cut with the restriction enzyme BsiWI, using the In-Fusion HD PCR cloning kit (Clontech). The nucleotide sequence of the light chain of human chimeric LN22R8 and the amino acid sequence of the light chain are shown in SEQ ID NO: 30 and SEQ ID NO: 31, respectively.

1)-12-5 Construction of human chimeric LN22R8 IgG1 heavy chain expression vector

Using the cDNA encoding the variable region of the LN22R8 heavy chain obtained in 1)-11-1-3 as a template, PCR was performed using primers designed using In-fusion selection to amplify the variable region encoding the heavy chain. cDNA DNA fragment. At the site where pCMA-G1 was cut with the restriction enzyme BlpI, an In-Fusion HD PCR cloning kit (Clontech) was used to insert the amplified DNA fragment to construct an IgG1 heavy chain expression vector of human chimeric LN22R8. The nucleotide sequence of the IgG1 type heavy chain of human chimeric LN22R8 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 32 and SEQ ID NO: 33, respectively.

1)-12-6 Construction of human chimeric LN22R8 IgG2 heavy chain expression vector

The cDNA encoding the variable region of the LN22R8 heavy chain obtained in Example 1)-11-1-3 was used as a template, and PCR was performed using the primers designed for in-fusion selection and cloning. The DNA fragments of cDNA in variable regions are amplified. At the site where pCMA-G2 was cut with the restriction enzyme BlpI, an In-Fusion HD PCR cloning kit (Clontech) was used to insert the amplified DNA fragment to construct an IgG2 heavy chain expression vector of human chimeric LN22R8. The nucleotide sequence of the IgG2 type heavy chain of human chimeric LN22R8 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 34 and SEQ ID NO: 35, respectively.

1)-12-7 Construction of human chimeric LN22R8 IgG4P heavy chain expression vector

A DNA fragment containing the DNA sequence encoding the amino acid sequence of the IgG4P heavy chain of human chimeric LN22R8 shown in Sequence ID No. 36 (GENEART) was synthesized. Using the synthesized DNA fragment, a human chimeric LN22R8 IgG4P heavy chain expression vector was constructed in the same manner as in Example 1)-12-2. The amino acid sequence of the IgG4P heavy chain of human chimeric LN22R8 is shown in Sequence ID No. 37.

1)-13 Production of 2P10F2 human chimeric antibody expression vector 1)-13-1 Construction of human chimeric and humanized IgG1LALA heavy chain expression vector pCMA-G1LALA

pCMA-G1LALA was constructed in the same manner as in Example 1)-12-2 using a DNA fragment containing the DNA sequence encoding the human heavy chain message sequence shown in Sequence ID No. 38 and the amino acid sequence of the human IgG1LALA constant region.

1)-13-2 Construction of human chimeric and humanized IgG4P heavy chain expression vector pCMA-G4P

pCMA-G4P was constructed in the same manner as in Example 1)-12-2 using a DNA fragment containing the DNA sequence encoding the human heavy chain message sequence shown in Sequence ID No. 39 and the amino acid sequence of the human IgG4P constant region.

1)-13-3 Construction of human chimeric 2P10F2 light chain expression vector

The cDNA encoding the variable region of the light chain of 2P10F2 obtained in Example 1)-11-2 was used as a template to construct the light chain expression of human chimeric 2P10F2 in the same manner as in Example 1)-12-4. carrier. The nucleotide sequence of the light chain of human chimeric 2P10F2 and the amino acid sequence of the light chain are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

1)-13-4 Construction of human chimeric 2P10F2 IgG1 LALA heavy chain expression vector

The cDNA encoding the variable region of the heavy chain of 2P10F2 obtained in Example 1)-11-2 was used as a template, and PCR was performed using primers designed for in-fusion selection. The amplification included the ability to encode the heavy chain. DNA fragment of the variable region cDNA. The human chimeric 2P10F2 IgG1LALA type heavy chain expression vector was constructed by inserting the amplified DNA fragment using the In-Fusion HD PCR cloning kit (Clontech) where pCMA-G1LALA was cut with the restriction enzyme B1pI. The nucleotide sequence of the IgG1 LALA type heavy chain of human chimeric 2P10F2 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

1)-13-5 Construction of human chimeric 2P10F2 IgG2 heavy chain expression vector

The cDNA encoding the variable region of the heavy chain of 2P10F2 obtained in Example 1)-11-2 was used as a template to construct human chimeric 2P10F2 IgG2 heavy chain in the same manner as in Example 1)-12-6. Chain expression vector. The nucleotide sequence of the IgG2 type heavy chain of human chimeric 2P10F2 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively.

1)-13-6 Construction of human chimeric 2P10F2 IgG4P heavy chain expression vector

The cDNA encoding the variable region of the heavy chain of 2P10F2 obtained in Example 1)-11-2 was used as a template, and PCR was performed using primers designed for in-fusion selection to amplify the DNA encoding the heavy chain. DNA fragment of the variable region cDNA. At the site where pCMA-G4P was cut with the restriction enzyme BlpI, an In-Fusion HD PCR cloning kit (Clontech) was used to insert the amplified DNA fragment to construct an IgG4P heavy chain expression vector of human chimeric 2P10F2. The nucleotide sequence of the IgG4P type heavy chain of human chimeric 2P10F2 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 46 and SEQ ID NO: 47, respectively.

1)-14 Production and preparation of human chimeric antibodies to LN22R8 and 2P10F2 1)-14-1 Production of human chimeric antibodies of LN22R8 and 2P10F2

FreeStyle 293F cells (Invitrogen) were subcultured and cultured according to the manual. 1.2×10 ⁹ FreeStyle 293F cells (Invitrogen) in the logarithmic growth phase were inoculated into 3L Fernbach Erlenmeyer Flask (CORNING), diluted with FreeStyle293 expression medium (Invitrogen) to prepare 2.0×10 ⁶ cells/ml. Add 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector and 1.8 mg of polyethyleneimine (Polyscience#24765) to 40 ml of Opti-Pro SFM medium (Invitrogen Company), stir slowly, and then place After 5 minutes, add to FreeStyle 293F cells. After culturing for 4 hours in a 37°C, 8% CO ₂ incubator with shaking at 90 rpm, 600 ml of EX-CELL VPRO medium (SAFC Biosciences), 18 mL of GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ultrafiltrate (GIBCO) were added. ), culture the culture supernatant obtained by shaking at 90 rpm for 7 days in a 37°C, 8% CO ₂ incubator, and filter it with a Disposable Capsule Filter (Advantec#CCS-045-E1H).

The human chimeric antibody of LN22R8 obtained by combining the IgG1 heavy chain expression vector of human chimeric LN22R8 and the light chain expression vector of human chimeric LN22R8 was named "LN22R8chIgG1". The human chimeric antibody of LN22R8 obtained by combining the IgG2 heavy chain expression vector of human chimeric LN22R8 and the light chain expression vector of human chimeric LN22R8 was named "LN22R8chIgG2". The human chimeric antibody of LN22R8 obtained by combining the IgG4P heavy chain expression vector of human chimeric LN22R8 and the light chain expression vector of human chimeric LN22R8 was named "LN22R8chIgG4P". The human chimeric antibody of L2P10F2 obtained by combining the IgG1LALA type heavy chain expression vector of human chimeric 2P10F2 and the light chain expression vector of human chimeric 2P10F2 was named "2P10F2chIgG1LALA". The human chimeric antibody of L2P10F2 obtained by combining the IgG2 heavy chain expression vector of human chimeric 2P10F2 and the light chain expression vector of human chimeric 2P10F2 was named "2P10F2chIgG2". The human chimeric antibody of L2P10F2 obtained by combining the IgG4P heavy chain expression vector of human chimeric 2P10F2 and the light chain expression vector of human chimeric 2P10F2 was named "2P10F2chIgG4P".

1)-14-2 Purification of human chimeric antibodies of LN22R8 and 2P10F2

From the culture supernatant obtained in Example 1)-14-1, the antibody was purified by the one-stage step of rProtein A affinity chromatography. The culture supernatant was supplied to a MabSelectSuRe-filled column (manufactured by GE Healthcare Bioscience) equilibrated with PBS, and then the column was washed with PBS at least twice the column capacity. Next, the solution was eluted with 2M arginine hydrochloride solution (pH 4.0), and the fraction containing the antibody was collected. This fraction was subjected to buffer replacement of HBSor (25mM histidine/5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using Centrifugal UF Filter Device VIVASPIN20 (fractionated molecular weight UF10K, Sartorius), and the IgG concentration was adjusted to 1 mg/mL or more. Finally, it was filtered with Minisart-Plus filter (Sartorius Company) and used as a purified sample.

1) ADCC activity of -15 human chimeric antibody

The ADCC activity of human chimeric antibodies was evaluated using human peripheral blood mononuclear cells (PBMC) as effector cells and human pancreatic cancer strain MIA PaCa-2 as ADCC target cells. MIA PaCa-2 cells labeled with radioisotope ⁵¹ Cr and mouse antibody (LN22R8), rat antibody (2P10F2), or human chimeric antibody (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P, 2P10F2chIgG1LALA, 2P10F2chIgG4P), at 0.5 or 5 μg / ml concentration, after treatment at 4°C for 30 minutes, PBMC isolated from human peripheral blood were added at a rate 20 times that of MIA PaCa-2 cells, and cultured at 37°C in the presence of 5% CO2 for 4 hours. The ⁵¹ Cr released in the supernatant was measured using TopCount NXT v2.53 to obtain the total release value. The measured value of ⁵¹ Cr released from MIA PaCa-2 cells labeled with ⁵¹ Cr and treated with Triton-100 was set as the maximum release value, and the measured value of ⁵¹ Cr released from the treatment of antibody-treated cells without adding PBMC was set as The spontaneous release value was calculated from the following formula and the specific release % was summarized in Figure 6. As a negative control sample, a sample treated with human IgG (hlgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was measured in the same manner and presented together. The measurement system was carried out three times, and the average value and standard deviation were calculated and presented together.

Specific release % = (total release - spontaneous release) / maximum release

Compared to human IgG (hIgG) and LN22R8 mouse antibodies, which did not show ADCC activity, LN22R8chIgG1 showed ADCC activity at 17.4% at 0.5 μg/ml and 18.1% at 5 μg/ml. The ADCC activities of LN22R8chIgG2 and LN22R8chIgG4P are lower than those of LN22R8chIgG1, even at 5 μg/ml, they are 3.0% and 2.2% respectively.

2P10F2 rat antibody showed ADCC activity at 4.8% at 0.5 μg/ml and 8.4% at 5 μg/ml. 2P10F2chIgG1LALA showed ADCC activity at 4.7% at 0.5 μg/ml and 2.9% at 5 μg/ml. 2P10F2chIgG4P showed lower ADCC activity than 2P10F2 rat antibody and 2P10F2chIgG1LALA, which was 3.4% at 0.5 μg/ml and 1.1% at 5 μg/ml. As reported in the literature (Bruggemann et al., J. Exp. Med., 1351-1361, 1987), human chimeric antibodies utilizing the IgG1 subtype showed the highest ADCC activity.

1) CDC activity of -16 human chimeric antibody

The complement-dependent cytocidal activity (CDC activity) of the anti-human CD147 antibody was evaluated using the human pancreatic cancer strain MIA PaCa-2 as a target cell. Commercially available rabbit complement (Low Tox-M Rabbit Complement, CEDARLANE LABORATORIES LIMITED, Cat. CL3051) was used as complement. As anti-human CD147 antibodies, mouse antibodies (LN22R8), rat antibodies (2P10F2), or human chimeric antibodies (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P, 2P10F2chIgG1LALA, 2P10F2chIgG4P) are used. Human IgG (hlgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was used as a negative control antibody for CDC activity. Treat the above antibodies at a concentration of 0, 0.1, 1 or 10 μg/ml at 4°C for 1 hour, add rabbit complement to a final concentration of 7.5%, and heat for 3 hours at 37°C in the presence of 5% CO2. Intracellular ATP contained in living cells was measured using CellTiter-Glo Lumimescent Cell Viability Assay (Promega, Cat. G7572). The luminescence signal obtained using CellTiter-Glo Lumimescent Cell Viability Assay was quantified using EnVision 2104 Multilabel Reader (Perkin Elmer). The measurement system was carried out three times, and the average value and standard deviation were calculated. The luminescent signal obtained from untreated cells was set as 100%, and the luminescent signal that was reduced in a complement-dependent manner with the antibody was regarded as CDC activity and is summarized in Figure 7 .

Concentration-dependent CDC activity compared to the negative control hIgG was only observed for mouse antibody (LN22R8) and rat antibody (2P10F2). In LN22R8, at 10 μg/ml, the viable cells were reduced to a maximum of 41.1%. In 2P10F2, at 10 μg/ml, viable cells were reduced to a maximum of 53.5%.

For human chimeric antibodies (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P, 2P10F2chIgG1LALA, 2P10F2chIgG4P), no clear CDC activity was observed relative to the negative control hIgG.

1) ADCP activity of -17 human chimeric antibody

It has been reported that human IgG antibodies exhibit cytocidal activity against cancer cells by inducing antibody-dependent monocyte-macrophage phagocytosis (ADCP) through interaction with mouse Fcγ receptors (Overdijk et al., Journal of Immunology, 1-9, 2012). The ADCP activity of human chimeric antibodies was evaluated using RAW264.7 (ATCC, TIB-71) as effector cells and human pancreatic cancer strain PANC-1 or MIA PaCa-2 as ADCP target cells. Add ADCP-labeled cells labeled with PKH67 Green Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (SIGMA, Cat.MINI67-1KIT) and human chimeric antibodies (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P) at a concentration of 20 μg/ml at 4°C. After 1 hour of treatment, RAW264.7 cells labeled with PKH26 Red Fluorescent Cell Linker Lit for General Cell Membrane Labeling (SIGMA, Cat. PKH26GL-1KT) were added with 5 times of ADCP-labeled cells and incubated at 37°C in the presence of 5% CO2. Warm for 3 hours. A flow cytometer (BD, CantoII) was used to measure the rate of PKH26-positive cells that migrated to PKH67 signal-positive cells through phagocytosis. As a negative control sample, a sample treated with human IgG (hlgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was measured in the same manner. The measurement was performed three times, and the average value and standard deviation were calculated. The results of PANC-1 in Figure 8(a) are shown in the results of MIA PaCa-2 in Figure 8(b).

When PANC-1 cells were used as ADCP targets, LN22R8chIgG1 was 9.2% and LN22R8chIgG4P was 9.0%, showing high ADCP activity compared to human IgG (5.5%). LN22R8chIgG2 was 5.9%, and ADCP activity was negative.

When MIA PaCa-2 cells were used as ADCP targets, the same tendency was shown. LN22R8chIgG1 was 6.6% and LN22R8chIgG4P was 6.1%, showing high ADCP activity compared to human IgG (3.6%). LN22R8chIgG2 was 3.6%, and ADCP activity was negative.

1) In vivo anti-tumor activity determination of -18 human chimeric antibody

5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 4 to 5-week-old female NOD-scid mice (NOD .CB17-Prkdc<scid>/J, purchased from Charles River, Japan, under the skin of the axilla. Based on the tumor volume, groups were implemented 5 to 7 days after transplantation, and the anti-human CD147 antibody LN22R8 mouse antibody (LN22R8) and three human chimeric antibodies (LN22R8chIgG1, LN22R8chIgG2, LN22R8chIgG4P) were administered at 1 mg/kg, 3 mg/kg, 10 mg/kg was administered intraperitoneally to cancer-bearing mice (n=5). Anti-human CD147 antibody 2P10F2 rat antibody (2P10F2) and two human chimeric antibodies (2P10F2chIgG2, 2P10F2chIgG4P) were administered into the abdominal cavity of cancer-bearing mice at 10 mg/kg (n=5~6). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figures 9-1(a)~(d) and Figures 9-2(e)~(g). For the changes in tumor volume in the figure, the mean value and standard error are recorded together.

LN22R8, mouse antibody, and three human chimeric antibodies also showed dose-dependent anti-tumor effects. In the group administered 10 mg/kg of human chimeric antibody LN22R8chIgG4P, complete tumor regression was observed in 5 out of 5 mice 18 days after transplantation. Even at the end of the experiment, 41 days after transplantation, no recurrence of tumors was seen. proliferation. In other LN22R8 antibody-administered groups, tumor repopulation was observed in some or all of the mice.

The anti-tumor effect was confirmed on 2P10F2, rat antibody, and two types of human chimeric antibodies. In the 2P10F2chIgG4P 10 mg/kg group, complete tumor regression was observed in 6 out of 6 mice 21 days after transplantation.

The mouse antibody LN22R8 and the rat antibody 2P10F2, which recognize the same epitope part of the anti-human CD147 antibody, are not only human chimeric antibodies chIgG1 that have effector functions of the mouse immune system that depend on ADCC activity, ADCP, CDC activity, etc. Or the human chimeric antibody chIgG4P with ADCP activity, because even the human chimeric antibody chIgG2, which shows almost no effector function, has more than 90% of the anti-tumor effect maintained, suggesting that it does not depend on mouse immunity, showing that by Anti-tumor effects of a novel mechanism of action on CD147.

1) Anti-tumor effect of -19 CD147 human chimeric antibody in NOG mice

In NOG (NOD/Shi-scid, IL-2Rγnull) mice, in NOD-scid mice lacking mouse T cells and B cells, IL-2 receptor γ, which shares a domain with the cytokine receptor, Mating with chain-knockout mice not only lacks mouse T and B cells, but also lacks NK cell and complement activity. It can be seen that the function of macrophages or dendritic cells is reduced, resulting in a state of extremely severe immunodeficiency (Ito, Blood, 3175- 3182, 2002). In the severely insufficient immune system of these mice, the anti-tumor effect of the CD147 antibody was verified using the subcutaneous transplantation model of MIA PaCa-2.

5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 7-week-old female NOG mice (NOD/Shi- scid, IL-2RγKO Jic, purchased from In-Vivo Science Company), subcutaneously in the axilla. Based on tumor volume, groups were divided into groups 6 days after transplantation, and anti-CD147 human chimeric antibody (LN22R8chIgG4P) was administered intraperitoneally into the abdominal cavity of cancer-bearing mice at 10 mg/kg (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 10. For the changes in tumor volume in the figure, the mean value and standard error are recorded together.

In the results of the human chimeric antibody LN22R8chIgG4P shown in Figure 10, the tumor growth inhibition rate 17 days after transplantation was 99% in the 10 mg/kg administration group, and complete tumor regression was observed in 3 out of 5 mice.

The anti-CD147 human chimeric antibody (LN22R8chIgG4P) shows strong anti-tumor effects on pancreatic cancer tumors formed in NOG mice that are deficient in T and B cells, as well as NK cells and complement activity, suggesting independence. The possibility of showing anti-tumor effects on immune cells in mice.

(Example 2) Preparation of monkey cross-ratio antibodies induced by immunization with CD147 protein

The anti-human CD147 antibody showing strong anti-tumor effect obtained in Example 1 did not show cross-activity against CD147 in mice, rats, and cynomolgus monkeys. Using the antibody obtained in Example 1, an attempt was made to obtain a CD147 antibody showing cross-activity with cynomolgus CD147.

2)-1 immunity

For immunization, WKY/Izm female rats (Japan SLC Corporation) were used. Recombinant Human BSG, a mixture of His tagged antigen protein (manufactured by CREATIVE BIOMART Co., Ltd.) and Freund's Complete Adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.) was administered to the lymph nodes and lymph nodes of the rat at the base of the tail. The spleen is used for fusion tumor production.

2)-2 Preparation of fusion tumor

Using LF301 Cell Fusion Unit (BEX Company), lymph node cells or spleen cells were electrically fused with mouse myeloma SP2/0-ag14 cells (ATCC, No. CRL-1581), and diluted in ClonaCell-HY Selection Medium D (StemCell Technologies). By recovering the fusion tumor community that emerged, a single fusion tumor was produced. Each recovered fusion tumor community is cultured, and the obtained fusion tumor culture supernatant is used to screen fusion tumors producing anti-CD147 antibodies.

2)-3 Antibody screening using flow cytometry

In order to select antibodies that bind to human cancer cells and show binding to human CD147 and cynomolgus monkey CD147 to generate fusion tumors, antibody binding screening using flow cytometry was performed. The CD147-positive human pancreas strain MIA PaCa-2 was used as human cancer cells. In the same manner as in Example 1)-8, CHO-K1 cells expressing human or cynomolgus CD147 (CHO-K1-hCD147v2, CHO-K1-cynoCD147) were used to bind to human or cynomolgus CD147. sexual confirmation. Add equal amounts of the fusion tumor culture supernatant to the suspension of MIA PaCa-2, CHO-K1-hCD147v2, and CHO-K1-cynoCD147, and react at 4°C for more than 1 hour. After washing, anti-rat IgG-PE (BD Biosciences, Cat. 550767) was used, and the binding of each antibody to the cells became detectable by fluorescence. The fluorescence signal of the cells was measured using a flow cytometer (CantoII, BD Bioscience), the ratio of the fluorescence signal to the negative control sample (cells to which fusion tumor culture medium was not added) was calculated, and a part of the results were sorted out. in Table 2.

Using a commercially available rat antibody isotyping kit (Bio-Rad Laboratories, RMT1), the isotypes (Isotypes) of the antibodies contained in the culture supernatants were determined and combined, as shown in Table 4.

2P10F2 obtained in Example 1 showed binding to MIA PaCa-2 and CHO-K1-hCD147v2, but showed no binding to CHO-K1-cynoCD147. rat_CD147_#84 (in this manual, it is also marked as r#84), rat_CD147_#131 (in this manual, it is also marked as r#131), rat_CD147_#110 (in this manual, it is also marked as r#131) In the case of r#110), rat_CD147_#101 (in this manual, the case is also marked as r#101), it shows binding to MIA PaCa-2, CHO-K1-hCD147v2, CHO-K1-cynoCD147, and is available Anti-human CD147 rat antibody showing cross-activity to cynomolgus monkeys.

2)-4 Preparation of rat monoclonal antibodies using low IgG serum

An anti-human CD147 monoclonal antibody system showing crossover to cynomolgus monkeys was purified from fusion tumor culture supernatants. First, the rat_CD147_#131 antibody-producing fusion tumor was grown to a sufficient amount using ClonaCell-HY Selection Medium E, and then the medium was exchanged with Hybridoma SFM (Life Technologies) supplemented with 20% Ultra Low IgG FBS (Life Technologies) , cultivate for 7 days. The culture supernatant was recovered and sterilized through a 0.45 μm filter.

2)-5 Preparation of rat monoclonal antibodies due to high-density culture

Rat_CD147_#84, rat_CD147_#101, and rat_CD147_#110 fusion tumors were cultured using a CL-1000 flask (Becton Dickinson Co., Ltd., Japan), and a fusion tumor culture supernatant containing a monoclonal antibody was prepared.

2) Purification of -6 monoclonal antibodies

From the culture supernatant prepared in Examples 2)-4 and Example 2)-5, the antibody was purified in the same manner as in Example 1)-6.

2)-7 Antibody screening resulting from in vivo anti-tumor activity assay

5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 5-week-old female NOD-scid mice (NOD.CB17 -Prkdc<scid>/J, purchased from Charles River, Japan, under the skin of the armpit. Based on the tumor volume, groups were divided into groups 6 to 8 days after transplantation. Cynomolgus monkey cross-type anti-CD147 rat antibodies #84, #101, or #110 were administered at 10 mg/kg on days 8 and 15 after transplantation. Intraperitoneal cavity of cancer-bearing mice (n=5). In the control group of mice, PBS was administered intraperitoneally in the same manner. After transplantation of cynomolgus cross-type anti-CD147 rat antibody #131, 10 mg/kg was administered intraperitoneally to cancer-bearing mice 6 days later (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figures 11(a) to (d). In the figure, the changes in tumor volume are combined with the mean and standard error. r#84, the tumor growth inhibition rate 28 days after transplantation was 95% in the 10 mg/kg group (Fig. 11(a)). For r#101, the tumor growth inhibition rate 15 days after transplantation was 37% in the 10 mg/kg group, but tumor re-proliferation was seen 28 days after transplantation (Fig. 11(b)). For r#110, the tumor proliferation inhibition rate in the 10 mg/kg group was 51% 15 days after transplantation, and tumor re-proliferation was seen 28 days after transplantation (Fig. 11(c)). r#131, the tumor growth inhibition rate 16 days after transplantation was 50% in the 10 mg/kg group (Fig. 11(d)). We obtained rat antibody r#84 that strongly inhibits tumor growth of MIA PaCa-2, and r#101, r#110, and r#131 that partially inhibited it.

The results are shown in Figures 11(a) to (d). For the changes in tumor volume in the figure, the mean value and standard error are recorded together. r#84, the tumor growth inhibition rate 28 days after transplantation was 95% in the 10 mg/kg group (Fig. 11(a)). For r#101, the tumor proliferation inhibition rate in the 10 mg/kg group was 37% 15 days after transplantation, and tumor re-proliferation was seen 28 days after transplantation (Fig. 11(b)). For r#110, the tumor proliferation inhibition rate in the 10 mg/kg group was 51% 15 days after transplantation, and tumor re-proliferation was seen 28 days after transplantation (Fig. 11(c)). For r#131, the tumor growth inhibition rate 16 days after transplantation was 50% in the 10 mg/kg group (Fig. 11(d)).

2)-8 epitope analysis: 2P10F2-competitive ELISA

For the purpose of analyzing the epitope of monkey cross-ratio CD147 antibody, the binding property of 2P10F2chIgG4P to CD147 recombinant protein was investigated by competitive ELISA. Human CD147-Fc fusion protein (Sino Biological Inc., 10186-H02H) was dissolved in PBS and adjusted to 20 μg/ml. 50 μl was added to a 96-well plate (Thermofisher, Cat. 43454) and stored at 4°C. Remove the protein solution, add 300 μl of PBS containing 1% BSA, and heat at room temperature for 1 hour. Add 25 μl of 20 or 60 μg/ml CD147 rat antibody r#84, r#101, r#110, r#131 or 2P10F2, or 2P10F2 containing 1% BSA diluted in PBS containing 1% BSA to a 96-well plate. PBS was used as a competing antibody and was added to a 96-well plate and incubated at room temperature for 2 hours. Add 25 μl of 20 ng/ml 2P10F2chIgG4P diluted in PBS containing 1% BSA to a 96-well plate and incubate at room temperature for 2 hours. Wash the 96 wells twice with PBS containing 0.05% Tween 20 (BIO RAD, Cat. 170-6531). Add 50 μl of Mouse monoclonal HP6025 Anti-Human IgG4 Fc (HRP) (Abcam, Cat. ab99823) diluted 2000-fold with PBS containing 1% BSA to a 96-well plate, and incubate at room temperature for 1 hour. Wash the 96 wells three times with PBS containing 0.05% Tween 20 (BIO RAD, Cat. 170-6531). Add 50 μl of Super AquaBlue ELISA Substrate (eBioscience Company, 00-4203-58) to the 96-well plate and warm at room temperature for 20 minutes. The absorbance at 405 nm of the 96-well plate was measured using EnVision 2104 Multilabel Reader (Perkin Elmer Company). Using the measured value of the well without competing antibody as a control, the absorbance decreased by the competing antibody was calculated in % and is shown in Figure 12 . The measurement was performed in 3 wells, and the average value is shown.

Similar to the 2P10F2 rat antibody, r#84, r#101, and r#131 inhibited the binding of 2P10F2chIgG4P by more than 90%, suggesting that the antibody recognition site is similar to that of 2P10F2. r#110 did not inhibit the binding of 2P10F2 rat antibody. Since the antibody recognition site is separated from 2P10F2 or the binding is weak, it is considered that there is a possibility that the binding of the 2P10F2 rat antibody cannot be inhibited.

(Example 3) Selection of monkey cross-ratio antibodies and production of human chimeric antibodies 3)-1 Determination of the selected nucleotide sequence of the cDNA encoding the variable region of the rat anti-CD147 antibody 3)-1-1 Determination of the nucleotide sequence of the cDNA encoding the variable region of rat_CD147_#84 antibody

It was carried out in the same manner as in Example 1)-11-2. The determined nucleotide sequence of the cDNA encoding the variable region of the light chain of the rat_CD147_#84 antibody is shown in SEQ ID NO: 48, and the amino acid sequence is shown in SEQ ID NO: 49. The nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 50, and the amino acid sequence is shown in SEQ ID NO: 51. CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_#84 antibody are shown in Sequence ID Nos. 52, 53 and 54, respectively. CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_#84 antibody are shown in Sequence ID Nos. 55, 56 and 57, respectively.

3)-1-2 Determination of the nucleotide sequence of the cDNA encoding the variable region of rat_CD147_#101 antibody

It was carried out in the same manner as in Example 1)-11-2. The nucleotide sequence of the cDNA encoding the variable region of the light chain of the determined rat_CD147_#101 antibody is shown in SEQ ID NO: 58, and the amino acid sequence is shown in SEQ ID NO: 59. The nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 60, and the amino acid sequence is shown in SEQ ID NO: 61. CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_#101 antibody are shown in Sequence ID Nos. 62, 63 and 64, respectively. CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_#101 antibody are shown in Sequence ID Nos. 65, 66 and 67, respectively.

3)-1-3 Determination of the nucleotide sequence of the cDNA encoding the variable region of rat_CD147_#110 antibody

It was carried out in the same manner as in Example 1)-11-2. The nucleotide sequence of the cDNA encoding the variable region of the light chain of the determined rat_CD147_#110 antibody is shown in SEQ ID NO: 68, and the amino acid sequence is shown in SEQ ID NO: 69. The nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 70, and the amino acid sequence is shown in SEQ ID NO: 71. CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_#110 antibody are shown in Sequence ID Nos. 72, 73 and 74, respectively. CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_#110 antibody are shown in Sequence ID Nos. 75, 76 and 77, respectively.

3)-1-4 Determination of the nucleotide sequence of the cDNA encoding the variable region of rat_CD147_#131 antibody

It was carried out in the same manner as in Example 1)-11-2. The nucleotide sequence of the cDNA encoding the variable region of the light chain of the determined rat_CD147_#131 antibody is shown in SEQ ID NO: 78, and the amino acid sequence is shown in SEQ ID NO: 79. The nucleotide sequence of the cDNA encoding the variable region of the heavy chain is shown in SEQ ID NO: 80, and the amino acid sequence is shown in SEQ ID NO: 81. CDRL1, CDRL2 and CDRL3 of the light chain variable region of the rat_CD147_#131 antibody are shown in Sequence ID Nos. 82, 83 and 84, respectively. CDRH1, CDRH2 and CDRH3 of the heavy chain variable region of the rat_CD147_#131 antibody are shown in Sequence ID Nos. 85, 86 and 87, respectively.

3)-2 Production of human chimeric antibody expression vector 3)-2-1 Production of human chimeric antibody expression vector rat_CD147_#84 3)-2-1-1 Construction of human chimeric and humanized IgG4PFALA heavy chain expression vector pCMA-G4PFALA

pCMA-G4PFALA was constructed using the same method as in Example 1)-12-2 using the DNA fragment shown in Sequence ID No. 88 and containing the DNA sequence encoding the human heavy chain message sequence and the amino acid sequence of the human IgG4PFALA constant region.

3)-2-1-2 Construction of human chimeric rat_CD147_#84 light chain expression vector

The cDNA encoding the variable region of the light chain of rat_CD147_#84 obtained in Example 3)-1-1 was used as a template to construct human chimeric rat_CD147_#84 in the same manner as in Example 1)-12-4. Light chain expression vector. The nucleotide sequence of the light chain of human chimeric rat_CD147_#84 and the amino acid sequence of the light chain are shown in SEQ ID NO: 89 and SEQ ID NO: 90, respectively.

3)-2-1-3 Construction of human chimeric rat_CD147_#84 IgG1 heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#84 obtained in Example 3)-1-1 as a template, human chimeric rat_CD147_#84 IgG1 was constructed in the same manner as in Example 1)-12-5. Type heavy chain expression vector. The nucleotide sequence of the IgG1 type heavy chain of human chimeric rat_CD147_#84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 91 and SEQ ID NO: 92, respectively.

3)-2-1-4 Construction of IgG2 heavy chain expression vector of human chimeric rat_CD147_#84

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#84 obtained in Example 3)-1-1 as a template, human chimeric rat_CD147_#84 was constructed in the same manner as in Example 1)-12-6. IgG2 heavy chain expression vector. The nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_#84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

3)-2-1-5 Construction of human chimeric rat_CD147_#84 IgG4P heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#84 obtained in Example 3)-1-1 as a template, human chimeric rat_CD147_#84 was constructed in the same manner as in Example 1)-13-6. IgG4P heavy chain expression vector. The nucleotide sequence of the IgG4P type heavy chain of human chimeric rat_CD147_#84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 95 and SEQ ID NO: 96, respectively.

3) Construction of the IgG1 LALA heavy chain expression vector of -2-1-6 human chimeric rat_CD147_#84

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#84 obtained in Example 3)-1-1 as a template, human chimeric rat_CD147_#84 was constructed in the same manner as in Example 1)-13-4. IgG1LALA type heavy chain expression vector. The nucleotide sequence of the IgG1 LALA type heavy chain of human chimeric rat_CD147_#84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively.

3)-Construction of IgG4PFALA heavy chain expression vector of human chimeric rat_CD147_#84

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#84 obtained in Example 3)-1-1 as a template, PCR was performed using primers designed for In-fusion selection, and amplification of the variable region encoding the heavy chain was carried out. DNA fragments of variable region cDNA. The IgG4PFALA type heavy chain expression of human chimeric rat_CD147_#84 was constructed by inserting the amplified DNA fragment using the In-Fusion HD PCR cloning kit (Clontech) where pCMA-G4PFALA was cut with restriction enzyme B1pI. carrier. The nucleotide sequence of the IgG4PFALA type heavy chain of human chimeric rat_CD147_#84 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively.

3)-2-2 Production of human chimeric antibody expression vector rat_CD147_#101 3)-2-2-1 Construction of human chimeric rat_CD147_#101 light chain expression vector

Using the cDNA encoding the variable region of the light chain of rat_CD147_#101 obtained in Example 3)-1-2 as a template, human chimeric rat_CD147_#101 was constructed in the same manner as in Example 1)-12-4. Light chain expression vector. The nucleotide sequence of the light chain of human chimeric rat_CD147_#101 and the amino acid sequence of the light chain are shown in SEQ ID NO: 101 and SEQ ID NO: 102, respectively.

3)-2-2-2 Construction of human chimeric rat_CD147_#101 IgG2 heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#101 obtained in Example 3)-1-2 as a template, human chimeric rat_CD147_#101 was constructed in the same manner as in Example 1)-12-6. IgG2 heavy chain expression vector. The nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_#101 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 103 and SEQ ID NO: 104, respectively.

3)-2-2-3 Construction of human chimeric rat_CD147_#101 IgG4P heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#101 obtained in Example 3)-1-2 as a template, human chimeric rat_CD147_#101 was constructed in the same manner as in Example 1)-13-6. IgG4P heavy chain expression vector. The nucleotide sequence of the IgG4P type heavy chain of human chimeric rat_CD147_#101 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 105 and SEQ ID NO: 106, respectively.

3) Construction of the IgG4PFALA type heavy chain expression vector of -2-2-4 human chimeric rat_CD147_#101

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#101 obtained in Example 3)-1-2 as a template, human chimeric rat_CD147_# was constructed in the same manner as in Example 3)-2-1-7. 101 IgG4PFALA heavy chain expression vector. The nucleotide sequence of the IgG4PFALA type heavy chain of human chimeric rat_CD147_#101 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 107 and SEQ ID NO: 108, respectively.

3)-2-3 Production of human chimeric antibody expression vector rat_CD147_#110 3)-2-3-1 Construction of human chimeric rat_CD147_#110 light chain expression vector

Using the cDNA encoding the variable region of the light chain of rat_CD147_#110 obtained in Example 3)-1-3 as a template, the human chimeric rat_CD147_#110 was constructed in the same manner as in Example 1)-12-4. Light chain expression vector. The nucleotide sequence of the light chain of human chimeric rat_CD147_#110 and the amino acid sequence of the light chain are shown in SEQ ID NO: 109 and SEQ ID NO: 110, respectively.

3)-2-3-2 Construction of human chimeric rat_CD147_#110 IgG2 heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#110 obtained in Example 3)-1-3 as a template, human chimeric rat_CD147_#110 was constructed in the same manner as in Example 1)-12-6. IgG2 heavy chain expression vector. The nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_#110 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 111 and SEQ ID NO: 112, respectively.

3)-2-3-3 Construction of human chimeric rat_CD147_#110 IgG4P heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#110 obtained in Example 3)-1-3 as a template, the human chimeric rat_CD147_#110 was constructed in the same manner as in Example 1)-13-6. IgG4P heavy chain expression vector. The nucleotide sequence of the IgG4P type heavy chain of human chimeric rat_CD147_#110 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 113 and SEQ ID NO: 114, respectively.

3)-Construction of IgG4PFALA heavy chain expression vector of human chimeric rat_CD147_#110

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#110 obtained in Example 3)-1-3 as a template, human chimeric rat_CD147_# was constructed in the same manner as in Example 3)-2-1-7. 110 IgG4PFALA heavy chain expression vector. The nucleotide sequence of the IgG4PFALA type heavy chain of human chimeric rat_CD147_#110 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 115 and SEQ ID NO: 116, respectively.

3)-2-4 Production of human chimeric antibody expression vector rat_CD147_#131 3)-2-4-1 Construction of human chimeric rat_CD147_#131 light chain expression vector

Using the cDNA encoding the variable region of the light chain of rat_CD147_#131 obtained in Example 3)-1-4 as a template, human chimeric rat_CD147_#131 was constructed in the same manner as in Example 1)-12-4. Light chain expression vector. The nucleotide sequence of the light chain of human chimeric rat_CD147_#131 and the amino acid sequence of the light chain are shown in SEQ ID NO: 117 and SEQ ID NO: 118, respectively.

3)-2-4-2 Construction of human chimeric rat_CD147_#131 IgG2 heavy chain expression vector

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#131 obtained in Example 3)-1-4 as a template, human chimeric rat_CD147_#131 was constructed in the same manner as in Example 1)-12-6. IgG2 heavy chain expression vector. The nucleotide sequence of the IgG2 type heavy chain of human chimeric rat_CD147_#131 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 119 and SEQ ID NO: 120, respectively.

3)-2-4-3 Construction of IgG4P heavy chain expression vector of human chimeric rat_CD147_#131

Using the cDNA encoding the variable region of the heavy chain of rat_CD147_#131 obtained in Example 3)-1-4 as a template, human chimeric rat_CD147_#131 was constructed in the same manner as in Example 1)-13-6. IgG4P heavy chain expression vector. The nucleotide sequence of the IgG4P type heavy chain of human chimeric rat_CD147_#131 and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 121 and SEQ ID NO: 122, respectively.

3)-3 Preparation of human chimeric antibodies 3)-3-1 Production of human chimeric antibodies to monkey cross-ratio antibodies

Produced by the same method as Example 1)-14-1.

The human chimeric antibody of rat_CD147_#84 obtained by combining the IgG1 heavy chain expression vector of human chimeric rat_CD147_#84 and the light chain expression vector of human chimeric rat_CD147_#84 was named "#84chIgG1". The human chimeric antibody of rat_CD147_#84 obtained by combining the IgG2 heavy chain expression vector of human chimeric rat_CD147_#84 and the light chain expression vector of human chimeric rat_CD147_#84 was named "#84chIgG2". The human chimeric antibody of rat_CD147_#84 obtained by combining the IgG4P heavy chain expression vector of human chimeric rat_CD147_#84 and the light chain expression vector of human chimeric rat_CD147_#84 was named "#84chIgG4P". The human chimeric antibody of rat_CD147_#84 obtained by combining the IgG1LALA type heavy chain expression vector of human chimeric rat_CD147_#84 and the light chain expression vector of human chimeric rat_CD147_#84 was named "#84chIgG1LALA". The human chimeric antibody of rat_CD147_#84 obtained by combining the IgG4PFALA type heavy chain expression vector of human chimeric rat_CD147_#84 and the light chain expression vector of human chimeric rat_CD147_#84 was named "#84chIgG4PFALA".

The human chimeric antibody of rat_CD147_#101 obtained by combining the IgG2 heavy chain expression vector of human chimeric rat_CD147_#101 and the light chain expression vector of human chimeric rat_CD147_#101 was named "#101chIgG2". The human chimeric antibody of rat_CD147_#101 obtained by combining the IgG4P heavy chain expression vector of human chimeric rat_CD147_#101 and the light chain expression vector of human chimeric rat_CD147_#101 was named "#101chIgG4P". The human chimeric antibody of rat_CD147_#101 obtained by combining the IgG4PFALA type heavy chain expression vector of human chimeric rat_CD147_#101 and the light chain expression vector of human chimeric rat_CD147_#101 was named "#101chIgG4PFALA".

The human chimeric antibody of rat_CD147_#110 obtained by combining the IgG2 type heavy chain expression vector of human chimeric rat_CD147_#110 and the light chain expression vector of human chimeric rat_CD147_#110 was named "#110chIgG2". The human chimeric antibody of rat_CD147_#110 obtained by combining the IgG4P heavy chain expression vector of human chimeric rat_CD147_#110 and the light chain expression vector of human chimeric rat_CD147_#110 was named "#110chIgG4P". The human chimeric antibody of rat_CD147_#110 obtained by combining the IgG4PFALA type heavy chain expression vector of human chimeric rat_CD147_#110 and the light chain expression vector of human chimeric rat_CD147_#110 was named "#110chIgG4PFALA".

The human chimeric antibody of rat_CD147_#131 obtained by combining the IgG2 type heavy chain expression vector of human chimeric rat_CD147_#131 and the light chain expression vector of human chimeric rat_CD147_#131 was named "#131chIgG2". The human chimeric antibody of rat_CD147_#131 obtained by combining the IgG4P heavy chain expression vector of human chimeric rat_CD147_#131 and the light chain expression vector of human chimeric rat_CD147_#131 was named "#131chIgG4P".

3)-3-2 Purification of human chimeric antibodies from monkey cross rat antibodies

The culture supernatant obtained from Example 3)-3-1 was purified in the same manner as in Example 1)-14-2.

(Example 4) In vivo anti-tumor activity of human chimeric antibodies

5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 5 to 6-week-old female NOD-scid mice ( NOD.CB17-Prkdc<scid>/J, purchased from Charles River, Japan, under the skin of the axilla. Based on the tumor volume, groups were implemented 5 to 6 days after transplantation. Cynomolgus monkey cross-type anti-CD147 human chimeric antibodies (#84chIgG1, #84chIgG1LALA, #84chIgG2, #84chIgG4P, #84chIgG4PFALA) were treated with 1, 3. Or 10 mg/kg was administered into the abdominal cavity of cancer-bearing mice (n=5). Cynomolgus monkey cross-type anti-CD147 human chimeric antibodies (#101chIgG2, #101chIgG4P, #101chIgG4PFALA, #110chIgG2, #110chIgG4P, #110chIgG4PFALA, #131chIgG2, #131chIgG4P) were administered at 3 or 10 mg/kg on the day after grouping. administered into the abdominal cavity of cancer-bearing mice (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 13-1(a)~(d), Figure 13-2(e)~(h), Figure 13-3(i)~(l), Figure 13-4(m) and (n) ). In the figure, the mean value and standard error are combined for changes in tumor volume.

Human chimeric antibody #84 shows dose-dependent anti-tumor effects of any IgG subtype. In the #84chIgG4P 10 mg/kg administration group, which was confirmed to have the strongest anti-tumor effect, complete tumor regression was confirmed in 5 out of 5 mice 20 days after transplantation.

Human chimeric antibody #101 shows dose-dependent anti-tumor effects of any IgG subtype. In the 10 mg/kg group of 101chIgG4P, which was confirmed to have the strongest anti-tumor effect, complete tumor regression was confirmed in 4 out of 5 mice 20 days after transplantation.

Human chimeric antibody #110 shows dose-dependent anti-tumor effects of either IgG subtype. In the 10 mg/kg group of 110chIgG4P, which was confirmed to have the strongest anti-tumor effect, complete tumor regression was confirmed in 3 out of 5 mice 22 days after transplantation.

Human chimeric antibody #131 is directed against the IgG4P subtype and exhibits dose-dependent anti-tumor effects. For #131chIgG2, there was no difference in the anti-tumor effect observed between the 3 mg/kg and 10 mg/kg administration groups.

Any human chimeric antibody is independent of the human IgG subtype of the antibody, and shows a tumor growth inhibitory effect of 68 to 100% at a dosage of 10 mg/kg. For the cynomolgus monkey cross-derived anti-human CD147 human chimeric antibodies #084, #101, #110, and #131, like the anti-human CD147 antibodies, LN22R8, or 2P10F2 obtained from cell-based immunization, they are not dependent on mouse immunity. system, suggesting that it exhibits anti-tumor effects through a novel mechanism of action on CD147.

(Example 5) Evaluation of CD147 binding properties of human chimeric antibodies

The dissociation constant of human CD147 of #84chIgG1, #84chIgG2, #84chIgG4P, #84chIgG1LALA, #84chIgG4PFALA, #101chIgG4P, and #110chIgG4P produced in Example 3)-3-1 was measured using Biacore T200 (manufactured by GE Healthcare Bioscience). A capture method is performed in which an anti-human IgG (Fc) antibody immobilized using the Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience) is captured using the antibody as a ligand, and the antigen is measured as the analyte. As the running buffer, HBS-EP+ (manufactured by GE Healthcare Bioscience) was used, and as the sensor wafer, CM5 (manufactured by GE Healthcare Bioscience) was used. After adding 1 μg/mL or 2 μg/mL human chimeric antibody to the chip at 10 μL/min for 60 seconds, a dilution series solution of the CD147 protein as the antigen (0.25~4 μg/mL for #131chIgG4P, and #84chIgG1, #84chIgG2, #84chIgG4P, #84chIgG1LALA, #84chIgG4PFALA, #101chIgG4P, #110chIgG4P (0.5~8μg/mL) were added at a flow rate of 30μL/min for 120 seconds, and then the dissociation phase was detected for 120 seconds, 300 seconds or 600 seconds. Among them, the CD147 protein was expressed in Escherichia coli, purified in two steps of Ni affinity and SEC, and then purified by tag cleavage. As a regeneration solution, 3M magnesium chloride (manufactured by GE Healthcare Bioscience) was added at a flow rate of 20 μL/min for 30 seconds. In the analysis of data, a 1:1 binding model was used to calculate the association rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD; KD=kd/ka). The results are shown in Table 5.

(Example 6) Preparation of humanized antibodies 6)-1 Design of humanized antibodies 6)-1-1 Molecular modeling of variable regions

As homology simulation, a well-known method (Methods in Enzymology, 203, 121-153, (1991)) was used. The commercially available protein three-dimensional structure analysis program Discovery Studio (manufactured by Dassault Systèmes) was used to search for proteins registered in a database of proteins with high sequence identity for variable regions (Nuc. Acids Res. 35, D301-D303 (2007)). Construct. The locked heavy chain, light chain and heavy chain-light chain interface structure were used as templates to create a three-dimensional model.

6)-1-2 Design method of humanized antibodies

Humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The framework region of rat_CD147_#84 is one of the human kappa chain subgroup 1 identified in KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) The consensus sequence and the consensus sequence of human gamma chain subgroup 3 have high identity, so they were each selected as the recipient of the light chain and heavy chain of rat_CD147_#84. The framework region of rat_CD147_#101 is based on the consensus sequence of the human kappa chain subgroup 1 determined by KABAT et al., the human gamma chain subgroup, and the human gamma chain IGHV3 specified by IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM®). -30*05 and IGHJ3*01 were each selected as the recipients of the light chain and heavy chain of rat_CD147_#101 due to their high identity. The framework region of rat_CD147_#110 is highly identical to IGKV1-39*01 and IGKJ4*01 of the human kappa chain specified by IMGT and to IGHV1-2*02 and IGHJ6*01 of the human gamma chain, so they are Each was selected as a recipient of the light and heavy chains of rat_CD147_#110. The framework region of rat_CD147_#131 has high similarity to IGKV1-39*01 and IGKJ2*01 of the human kappa chain specified by IMGT, and IGHV3-30*05 and IGHJ6*01 of the human gamma chain, so each was selected As a recipient of the light and heavy chains of rat_CD147_#131. The donor residues that should be transferred to the acceptor are selected by analyzing the three-dimensional model with reference to the standards given by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)), etc. .

6)-1-3 Humanization of rat_CD147_#84 heavy chain

For the variable region of rat_CD147_#84 heavy chain, the variable region of humanized antibody heavy chain #84H1h was designed by converting the amino acid residues of the acceptor shown in Figure 14(a).

Based on the designed variable regions, humanized antibody heavy chains were designed that connected the constant regions of the gamma chains of human IgG2 and IgG4P, and were named #84H1hIgG2 and #84H1hIgG4P respectively. The full-length amino acid sequence of #84H1hIgG2 is recorded in SEQ ID NO: 123. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 123 is shown in SEQ ID NO: 124. The full-length amino acid sequence of #84H1hIgG4P is recorded in Sequence ID No. 125. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 125 is described in SEQ ID NO: 126.

6)-1-4 Humanization of rat_CD147_#84 light chain

For the variable region of the rat_CD147_#84 light chain, the variable region of the humanized antibody light chain #84L2h was designed by moving in the amino acid residues of the acceptor shown in Figure 14(b).

Based on the designed variable region, a humanized antibody light chain connected to the human kappa chain constant region was designed and named #84L2h. The full-length amino acid sequence of the light chain of #84L2h is recorded in Sequence ID No. 127. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 127 is described in SEQ ID NO: 128.

6)-1-5 #84 Humanized Antibody

The humanized antibody #84H1L2hIgG2 was designed by combining the heavy chain #84H1hIgG2 and light chain #84L2h designed above. Furthermore, heavy chain #84H1hIgG4P and light chain #84L2h were combined to design humanized antibody #84H1L2hIgG4P.

6)-1-6 rat_CD147_#101 heavy chain humanization

For the variable region of rat_CD147_#101 heavy chain, the variable region of humanized antibody heavy chain #101H1h was designed by moving the amino acid residues of the acceptor shown in Figure 15(a).

Based on the designed variable regions, humanized antibody heavy chains were designed that connected the gamma chain constant regions of human IgG2 and IgG4P, and were named #101H1hIgG2 and #101H1hIgG4P respectively. The full-length amino acid sequence of #101H1hIgG2 is shown in SEQ ID NO: 129. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129 is described in SEQ ID NO: 130. The full-length amino acid sequence of #101H1hIgG4P is recorded in SEQ ID NO: 131. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 131 is described in SEQ ID NO: 132.

6)-1-7 Humanization of rat_CD147_#101 light chain

For the variable region of the rat_CD147_#101 light chain, the variable region of the humanized antibody light chain #101L2h was designed by moving the amino acid residues of the acceptor shown in Figure 15(b).

Based on the designed variable region, a humanized antibody light chain connected to the human kappa chain constant region was designed and named #101L2h. The full-length amino acid sequence of the light chain of #101L2h is recorded in Sequence ID No. 133. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 133 is described in SEQ ID NO: 134.

6)-1-8 #101 Humanized Antibody

The humanized antibody #101H1L2hIgG2 was designed by combining the heavy chain #101H1hIgG2 and light chain #101L2h designed above. Furthermore, heavy chain #101H1hIgG4P and light chain #101L2h were combined to design humanized antibody #101H1L2hIgG4P.

6)-1-9 rat_CD147_#110 heavy chain humanization

For the variable region of rat_CD147_#110 heavy chain, the variable regions of humanized antibody heavy chains #110H1h and #110H13h were designed by moving the amino acid residues of the acceptors shown in Figure 16(a) and (b). .

Based on the designed variable region, humanized antibody heavy chains connected to the gamma chain constant region of IgG4P were designed and named #110H1hIgG4P and #110H13hIgG4P respectively. The full-length amino acid sequence of #110H1hIgG4P is described in SEQ ID NO: 135, and the full-length amino acid sequence of #110H13hIgG4P is described in SEQ ID NO: 147. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 135 is described in SEQ ID NO. 136, and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 147 is described in SEQ ID NO. 148.

6)-1-10 rat_CD147_#110 light chain humanization

For the variable region of the rat_CD147_#110 light chain, humanized antibody light chains #110L4h and #110L2h were designed by moving the amino acid residues of the acceptors shown in Figure 16(c), (d), and (e). , variable area of #110L12h.

Based on the designed variable region, humanized antibody light chains connected to the human kappa chain constant region were designed and named #110L4h, #110L2h, and #110L12h respectively. The full-length amino acid sequence of the light chain of #110L4h is recorded in SEQ ID NO. 137, the full-length amino acid sequence of the light chain of #110L2h is recorded in SEQ ID NO. 149, and the full-length amino acid sequence of the light chain of #110L12h is recorded in SEQ ID NO. Identification number 151. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 137 is described in SEQ ID NO. 138, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 149 is described in SEQ ID NO. 150, and the coding sequence ID The nucleotide sequence of the amino acid sequence No. 151 is recorded in Sequence ID No. 152.

6)-1-11 #110 Humanized Antibody

The humanized antibody #110H1L4hIgG4P was designed by combining the heavy chain #110H1hIgG4P and light chain #110L4h designed above. Furthermore, heavy chain #110H13hIgG4P and light chain #110L2h were combined to design humanized antibody #110H13L2hIgG4P. Heavy chain #110H13hIgG4P and light chain #110L12h were combined to design humanized antibody #110H13L12hIgG4P.

6)-1-12 Humanization of rat_CD147_#131 heavy chain

For the variable region of rat_CD147_#131 heavy chain, the variable region of humanized antibody heavy chain #131H2h was designed by moving the amino acid residues of the acceptor shown in Figure 17(a).

Based on the designed variable region, a humanized antibody heavy chain was designed that connected the constant region of the gamma chain of human IgG2 and was named #131H2hIgG2. The full-length amino acid sequence of #131H2hIgG2 is recorded in SEQ ID NO: 139. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 is described in SEQ ID NO: 140.

6)-1-13 Humanization of rat_CD147_#131 light chain

For the variable region of rat_CD147_#131 light chain, the variable region of humanized antibody light chain #131L2h was designed by moving the amino acid residues of the acceptor shown in Figure 17(b).

Based on the designed variable region, a humanized antibody light chain was designed that connected the constant region of the human kappa chain and was named #131L2h. The full-length amino acid sequence of the light chain of #131L2h is recorded in Sequence ID No. 141. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 141 is described in SEQ ID NO: 142.

6)-1-14 #131 Humanized Antibody

The humanized antibody #131H2L2hIgG2 was designed by combining the heavy chain #131H2hIgG2 and light chain #131L2h designed above.

6)-2 Construction of humanized antibody light chain expression vector 6)-2-1 Construction of #84L2h expression vector

The DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of #84L2h represented by SEQ ID NO: 128 (GENEART Company) was synthesized. Using the In-Fusion HD PCR cloning kit (Clontech), the #84L2h expression vector was constructed by inserting the synthetic DNA fragment into pCMA-LK at the site cut off by the restriction enzyme BsiWI.

6)-2-2 Construction of #101L2h expression vector

The DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of #101L2h represented by SEQ ID NO. 134 was synthesized (GENEART Company). #101L2h expression vector was constructed in the same manner as in Example 6)-2-1.

6)-2-3 Construction of #110L4h, #110L2h and #110L12h expression vectors

DNA fragments represented by nucleotide numbers 37 to 402 of the nucleotide sequences of #110L4h, #110L2h and #110L12h represented by SEQ ID NO: 138, SEQ ID NO: 150 and SEQ ID NO: 152 were synthesized (GENEART Company). Expression vectors #110L4h, #110L2h and #110L12h were constructed in the same manner as in Example 6)-2-1.

6)-2-4 Construction of #131L2h expression vector

A DNA fragment represented by nucleotide numbers 37 to 402 of the nucleotide sequence of #131L2h represented by SEQ ID NO. 142 was synthesized (GENEART Company). #131L2h expression vector was constructed in the same manner as in Example 6)-2-1.

6)-3 Construction of humanized antibody heavy chain expression vector 6)-3-1 Construction of #84H1hIgG2 expression vector

The DNA fragment represented by nucleotide numbers 36 to 437 of the nucleotide sequence of #84H1hIgG2 represented by SEQ ID NO. 124 (GENEART Company) was synthesized. Using the In-Fusion HD PCR cloning kit (Clontech), the #84H1hIgG2 expression vector was constructed by inserting pCMA-G2 into the synthetic DNA fragment at the site cut off by restriction enzyme B1pI.

6)-3-2 Construction of #84H1hIgG4P expression vector

The DNA fragment represented by nucleotide numbers 36 to 437 of the nucleotide sequence of #84H1hIgG4P represented by SEQ ID NO. 126 was synthesized (GENEART Company). Using the In-Fusion HD PCR cloning kit (Clontech), the #84H1hIgG4P expression vector was constructed by inserting pCMA-G4P into the synthetic DNA fragment at the site cut off by restriction enzyme B1pI.

6)-3-3 Construction of #101H1hIgG2 expression vector

A DNA fragment represented by nucleotide numbers 36 to 428 of the nucleotide sequence of 101H1hIgG2 represented by SEQ ID NO: 130 (GENEART Company) was synthesized. #101H1hIgG2 expression vector was constructed in the same manner as in Example 6)-3-1.

6)-3-4 Construction of #101H1hIgG4P expression vector

A DNA fragment represented by nucleotide numbers 36 to 428 of the nucleotide sequence of 101H1hIgG4P represented by SEQ ID NO. 132 was synthesized (GENEART Company). #101H1hIgG4P expression vector was constructed in the same manner as in Example 6)-3-2.

6)-3-5 Construction of #110H1hIgG4P and #110H13hIgG4P expression vectors

DNA fragments represented by nucleotide numbers 36 to 425 of the nucleotide sequences of 110H1hIgG4P and #110H13hIgG4P represented by SEQ ID NO: 136 and SEQ ID NO: 148 were synthesized (GENEART Company). #110H1hIgG4P and #110H13hIgG4P expression vectors were constructed in the same manner as in Example 6)-3-2.

6)-3-6 Construction of #131H2hIgG2 expression vector

A DNA fragment represented by nucleotide numbers 36 to 431 of the 131H2hIgG2 nucleotide sequence represented by SEQ ID NO: 140 (GENEART Company) was synthesized. #131H2hIgG2 expression vector was constructed in the same manner as in Example 6)-3-1.

6)-4 Preparation of humanized antibodies 6)-4-1 Production of humanized antibodies

Produced by the same method as Example 1)-14-1. By corresponding to the combination of H chain and L chain shown in Example 6)-1-5, Example 6)-1-8, Example 6)-1-11, and Example 6)-1-14 The combination of H chain expression vector and L chain expression vector can obtain various humanized antibodies.

6)-4-2 First-stage purification of humanized antibodies

The antibody was purified from the culture supernatant obtained in Example 6)-4-1 by a one-stage step of rProtein A affinity chromatography. After the culture supernatant was applied to a column filled with PBS-equilibrated MabSelectSuRe (manufactured by GE Healthcare Bioscience), the column was washed with PBS at least twice the column capacity. Next, the solution was dissolved with 2M spermine hydrochloride solution (pH 4.0), and the fraction containing the antibody was collected. This fraction was subjected to buffer replacement of PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using Centrifugal UF Filter Device VIVASPIN20 (fractionated molecular weight UF10K, Sartorius), and the IgG concentration was adjusted to 1 mg/mL or more. Finally, it was filtered with Minisart-Plus filter (Sartorius Company) and used as a purified sample.

6)-4-3 2-stage purification of humanized antibodies

The culture supernatant obtained in Example 6)-4-1 was purified through a two-stage process of rProtein A affinity chromatography and ceramic hydroxy apatite. The culture supernatant was applied to a column filled with PBS-equilibrated MabSelectSuRe (manufactured by GE Healthcare Bioscience), and then the column was washed with PBS at least twice the column capacity. Next, the antibody was eluted with 2M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was replaced with PBS buffer by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), diluted 5 times with 5mM sodium phosphate/50mM MES/pH7.0 buffer, and then applied In a ceramic hydroxyapatite column (Bio-Scale CHT Type-1 Hydroxyapatite Column, Bio-Rad, Japan) equilibrated with a buffer of 5mM NaPi/50mM MES/30mM NaCl/pH7.0. Linear concentration gradient elution using sodium chloride was performed, and the antibody-containing fractions were collected. This fraction was subjected to buffer replacement of HBSor (25mM histidine/5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated using Centrifugal UF Filter Device VIVASPIN20 (fractionated molecular weight UF10K, Sartorius), and the IgG concentration was adjusted to 25 mg/mL. Finally, it was filtered with Minisart-Plus filter (Sartorius Company) and used as a purified sample.

(Example 7) Activity measurement of humanized antibodies 7)-1 Evaluation of CD147 antibody binding of humanized antibodies

The dissociation constant measurement system of humanized anti-human CD147 antibodies #84H1L2hIgG2, #84H1L2hIgG4P, #101H1L2hIgG2, #101H1L2hIgG4P, #110H1L4hIgG4P and #131H2L2hIgG2 prepared in Example 6)-4-3 and human CD147 was used using Biacore T200 (GE Healthcare Bioscience (manufactured by GE Healthcare Bioscience), the antibody is captured as a ligand on an anti-human IgG (Fc) antibody immobilized using the Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience), and the antigen is measured as the analyte. Law. HBS-EP+ (manufactured by GE Healthcare Bioscience) was used as the running buffer, and CM5 (manufactured by GE Healthcare Bioscience) was used as the sensor chip. 1 μg/mL humanized antibody was added to the chip at 10 μL/min for 60 seconds. Finally, the dilution series solution of the antigen used in Example 5) (for #101H1L2hIgG2 and #101H1L2hIgG4P, it is 0.0625~1 μg/mL; for #84H1L2hIgG2, #84H1L2hIgG4P, #110H1L4hIgG4P, #110H13L2hIgG4P, #1 10H13L12hIgG4P and #131H2L2hIgG2, are 0.25~4μg/mL), add 120 seconds at a flow rate of 30μL/min, and then monitor the dissociation phase for 300 seconds. A regeneration solution of 3M magnesium chloride (manufactured by GE Healthcare Bioscience) was added at a flow rate of 20 μL/min for 30 seconds. In the analysis of data, a 1:1 binding model was used to calculate the association rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD; KD=kd/ka).

The results are shown in Table 6.

7)-2 Anti-tumor effect of humanized CD147 antibody in pancreatic cancer-bearing cancer model

5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 4-week-old female NOD-scid mice (NOD. CB17-Prkdc<scid>/J, purchased from Charles River, Japan, under the skin of the axilla. Grouping was performed based on tumor volume, and humanized CD147 antibodies (#84H1L2hIgG2, #84H1L2Ig4P, #101H1L2hIg2, #101H1L2hIg4P, # prepared in Example 6)-4-2 were administered at 3 mg/kg and 10 mg/kg 7 days after transplantation. 110H1L4hIg4P, #131H2L2hIg2) in the abdominal cavity of cancer-bearing mice (n=5). On days 7 and 14 after transplantation, gemcitabine (purchased from Eli Lilly, Japan), a therapeutic drug for pancreatic cancer, was administered at 400 mg/k into the abdominal cavity of cancer-bearing mice (n=5) as a control drug. The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

The results are shown in Figures 18(a) to (d). In the figure, the mean value and standard error are combined for changes in tumor volume.

The tumor growth inhibition rate of the control drug was 71%, and no tumor disappearance was observed. In the 3 or 10 mg/kg administration group, either humanized CD147 antibody showed a superior anti-tumor effect than gemcitabine. A strong anti-tumor effect accompanied by tumor disappearance was confirmed in the 10 mg/kg administration group of #84H1L2hIg4P, #101H1L2hIg2, #101H1L2hIg4P, and #110H1L4hIg4P.

(Example 8) Activation of p38MAPK signal in vitro by CD147 antibody 8)-1 Induction of p38MAPK phosphorylation by anti-human CD147 human chimeric antibody in pancreatic cancer cell line PANC-1

It has been reported that CD147 promotes the phosphorylation of p38MAPK through activation (Lim et al., FEBS Letters, 88-92, 1998) (Li et al., al. J. Hepatology, 1378-1389, 2015). In order to investigate the effect of anti-CD147, which shows anti-tumor effect, on p38MAPK signaling, PANC-1 cells were treated with 10 μg/ml of CD147 human chimeric antibody LN22R8chIgG4P for 15 minutes. Simple Western assays (Protein Simple Japan) were used to phosphorylate p38MAPK. Co., Ltd., Wes). PANC-1 cells as a control sample were similarly treated with human IgG (Jackson ImmunoResearch, 009-000-003) 10 μg/ml and subjected to analysis. For the detection of p38MAPK, p38 MAPK rabbit mAb (Cell signaling technology company, Cat.#9212) was used. For the detection of phosphorylated p38MAPK, P-p38 MAPK(T180/Y182)(D3F9)XP rabbit mAb (Cell signaling technology company, #4511S) was used. The ratio of the detected phosphorylated p38MAPK signal to the p38MAPK signal is shown in Figure 19 . Antibody-treated samples were divided into 2 fractions, and the average values are shown in the graph.

In the LN22R8chIgG4P antibody-treated group, an increase in phosphorylated MAPK signals was observed.

8)-2 Phosphorylation of HSP27, a downstream molecule of p38 signaling by CD147 antibody in vitro

It has been reported that p38MAPK phosphorylates HSP27 through activation (Landry et al., Biochem. Cell Biol., 703-707, 1995). In order to confirm that the phosphorylation of p38 induced by the CD147 human chimeric antibody LN22R8 actually promotes p38 signal activation, samples treated with the antibody in 8)-1 were evaluated using Simple Western assays (Protein Simple Japan Co., Ltd., Wes). Phosphorylation. For the detection of HSP27, HSP27 antibody (R&D Systems, Cat. AF15801) was used. For the detection of phosphorylated HSP27, Phospho-HSP27 (Ser82) antibody (Cell signaling technology company, 2401S) or Anti-HSP27 (phospho Ser15) antibody (Abcam company, Cat. ab76313) is used.

The ratio of the Ser82 or Ser15 phosphorylated HSP27 signal to the detected HSP27 signal is shown in Figure 20(a) or Figure 20(b) respectively. Antibody-treated samples were carried out in duplicate, and the average values are shown in the figure.

In the LN22R8chIgG4P antibody-treated group, an increase in phosphorylated HSP27 signal was observed. Since the phosphorylation of p38 induced by the CD147 human chimeric antibody induces the phosphorylation activation of downstream HSP27, it was determined that the anti-human CD147 human chimeric antibody LN22R8 IgG4P induces the activation of the p38MAPK signal. Furthermore, the activation of p38MAPK signal was similarly confirmed in the pancreatic cancer cell MIA PaCa-2.

(Example 9) Activation of p38 signaling in tumors by CD147 antibody

Whether the activation of p38MAPK signaling caused by the CD147 antibody observed in vitro also occurs in tumors formed under the skin of mice was investigated using MIA PaCa-2 mouse subcutaneous tumors. 5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 5-week-old female NOD-scid mice (NOD. CB17-Prkdc<scid>/J, purchased from Charles River, Japan, under the skin of the axilla. Based on tumor volume, groups were implemented 5 days after transplantation. On the day after grouping, anti-human CD147 human chimeric antibody LN22R8chIgG4P was administered intraperitoneally into the abdominal cavity of cancer-bearing mice at 10 mg/kg. Tumor materials were collected 6, 24, 48, and 72 hours after antibody administration, frozen in dry ice, and then cryopreserved. For sample preparation of self-frozen tumor tissue (n=3), gentleMACS Octo Dissociator with Heaters (Miltenyi Company) was used. The prepared tumor lysate (lysate) was analyzed using Simple Western assays (Protein Simple Japan Co., Ltd., Wes).

The average value of the detected p38MAPK signal is shown in Figure 21(a) together with the standard error. The average value of the detected phosphorylated p38MAPK signal is shown in Figure 21(b) together with the standard error. The average value of the ratio of phosphorylated p38MAPK signal to p38MAPK signal is shown in Figure 21(c) together with the standard error. LN22R8chIgG4P increased the rate of phosphorylated P38MAPK from 6 hours to 72 hours after administration. By increasing p38MAPK from 24 hours to 72 hours, the signal of phosphorylated p38MAPK decreased reaching its peak at 6 hours.

In subcutaneous tumors of mice, an increase in p38 phosphorylation and an increase in HSP27 phosphorylation due to administration of the CD147 chimeric antibody were also observed, indicating that activation of p38 signaling occurs.

(Example 10) Induction of CXCL8, a molecule downstream of p38MAPK

Induction of mRNA stabilization by CXCL8 through activation of p38MAPK has been reported (Hoffmann et al., J. Leukoc. Biol., 847-855, 2002), activation of SMAD3/4 signaling (Leovonen et al. , PLOS ONE, e57474, 2013). Whether CXCL8 gene expression is induced in MIA PaCa-2 tumors after antibody administration was investigated by quantitative PCR using RNA extracted from mouse subcutaneous tumors after antibody administration. Similarly, the RHOB gene, which has been reported to be induced by activation of SMAD signaling (Vasilaki et al., FASEB Journal, 891-905, 2010), was also investigated for changes caused by administration of the CD147 antibody. The expression of importin (ipo8) gene and TATA box binding protein (tbp) gene as internal control genes were measured. The anti-human CD147 human chimeric antibody was administered, and MIA PaCa-2 tumors were collected 72 hours after treatment with RNA later (QIAGEN, Cat. 76104), and RNeasy Mini Kit (250) (QIAGEN, Cat. .74106), extract RNA. As the anti-human CD147 human chimeric antibody, LN22R8chIgG1, LN22R8chIgG2 or LN22R8chIgG4P prepared in Example 1)-14 was used. For quantitative RT-PCR, EXPRESS One-Step SuperScript qRT-PCR kit Universal (Thermofisher Scientific, Cat.11781-01K) was used. As a gene quantitative probe, importin (ipo8) (Thermo Fisher Scientific, Cat.Hs00183533_m1) was used. , TATA box binding protein (tbp) (Thermo Fisher Scientific, Cat.Hs00427621_m1), rashomolog family member B (rhoB) (Thermo Fisher Scientific, Cat.hs00269660_s1), interleukin 8 (Thermo Fisher Scientific, Cat.Hs00174103_m1). ABIPrism7500 (Applied Biosystems) was used to measure the increase in gene-specific fluorescent signal accompanying the PCR reaction.

In Figure 22(a), the average value (n=3) of the expression ratio of the ipo8/tbp gene is presented together with the standard deviation.

In Figure 22(b), the average value (n=3) of the expression ratio of the cxcl8/tbp gene is presented together with the standard deviation.

In Figure 22(c), the average value (n=3) of the expression ratio of the rhoB/tbp gene is presented together with the standard deviation.

After administration of LN22R8chIgG1, LN22R8chIgG2 or LN22R8chIgG4P, the expression of the ipo8 gene remained unchanged, but induction of the expression of cxcl8 and rhoB in the anti-human CD147 human chimeric antibody-administered group was confirmed. Based on the strength of the anti-tumor effect of the human chimeric antibodies shown in Examples 1) to 18, the expression induction of cxcl8 and rhoB suggests that the expression induction of the two genes may be related to the anti-tumor effect.

(Example 11) Induction of cxcl8 and rhoB genes in enhancing the anti-tumor effect by converting the anti-human CD147 rat antibody into a human chimeric antibody

The anti-human CD147 rat antibody rat_CD147_#110 has an enhanced anti-tumor effect by human chimerization as shown in Example 2 and Example 7. The induction of cxcl8 and rhoB observed in tumors after administration of human chimeric antibodies LN22R8chIgG1, LN22R8chIgG2, or LN22R8chIgG4P was compared with rat_CD147_#110 and chimeric antibody #110chIgG4P in the same manner as 14)-2.

In Figure 23(a), the average value (n=3) and the standard deviation of the expression ratio of the ipo8/tbp gene are combined and shown.

In Figure 23(b), the average value (n=3) and the standard deviation of the expression ratio of the cxcl8/tbp gene are combined and shown.

In Figure 23(c), the average value (n=3) and the standard deviation of the expression ratio of the rhoB/tbp gene are combined and shown.

The expression of the ipo8 gene was not changed by antibody administration. The expression of cxcl8 gene and rhoB gene was not changed by the administration of rat_CD147_#110 as in the antibody-unadministered group, but the induction of cxcl8 and rhoB by #110chIgG4P was confirmed. It is suggested that the induction of the two genes is a parameter related to the anti-tumor effect caused by CD147 antibodies.

(Example 12) Evaluation of SMAD4-negative pancreatic cancer-bearing cancer model

As one of the molecules important in the activation of SMAD signal, the transcription factor SMAD4 is known (Zang, et al., Current Biology, 270-276, 1997). It is known that in some pancreatic cancers, SMAD signaling is partially blocked by genetic defects in SMAD4 (Hahn, et al., Science, 350-353, 1996). It was investigated whether the CD147 antibody showed anti-tumor effect on the pancreatic cancer cell line BxPC-3, which is SMAD4-deficient and has partially blocked SMAD signaling (Yasutome et al., Clin. Exp. Metastasis, 461-473, 2005). 2.5×10 ⁶ cells of the human pancreatic cell line BxPC-3 (ATCC, Cat. CRL-1687) were suspended in PBS containing 100% Matrigel (Corning Company, Cat. 354234), and transplanted into BALB/C of 6-week-old females. subcutaneously in the axilla of c-nu mice (CAnN.Cg-Foxn1nu/CrlCrli, purchased from Charles River, Japan). Based on the tumor volume, groups were implemented 8 days after transplantation. On days 8, 15, and 22 after transplantation, mouse anti-human CD147 antibody LN22R8R8 or rat anti-human CD147 antibody 2P10F2 was administered to the tumor-bearing mice at 10 mg/kg. Intraperitoneal (n=5). On days 8, 15, and 22 after transplantation, gemcitabine (Gemzar (registered trademark), Eli Lilly Co., Ltd., Japan), a therapeutic drug for pancreatic cancer, was administered as a control drug at 200 mg/kg into the tail vein of the cancer-bearing mice. Within (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

Shown in Figure 24. In the figure, the mean value and standard error are combined for changes in tumor volume. The BxPC-3 tumor line is resistant to LN22R8 antibody, 2P10F2 antibody and gemcitabine.

(Example 13) Acquisition of CD147 antibody sensitivity by introduction of SMAD4

It has been reported that SMAD signaling is restored by introducing SMAD4 into multiple pancreatic cancer cell lines. Through the restored SMAD signal caused by the introduction of SMAD4, it was investigated whether the sensitivity of CD147 antibody is increased.

13)-1 Preparation of SMAD4 stability expressing cells

The Retro-X ^TM Q vector kit was used to create the SMAD4 stable expression strain of BxPC-3. As a retroviral vector (TAKARA BIO, Retro-X ^TM Q Vector Set, Cat. 631516), the synthetic human SMAD4 gene is introduced into the selection site of pQCXIP included in the set, and reverse transcription is expressed as SMAD4 Viral vectors. Retro-X Universal Packaging System (TAKARA BIO Company, Cat. 631530) was used to introduce the SMAD4-expressing retroviral vector into BxPC-3, and puromycin (TAKARA BIO Company, Cat. 631306) was used. Upon viral infection, the retrovirus is incorporated into the chromosome and puromycin-resistant, SMAD4-positive BxPC-3 cells are selected as SMAD4-positive BxPC-3 cells, BxPC-3-SMAD4. In the same manner, the retroviral vector pQCXIP was infected, and puromycin-resistant BxPC-3 cells were used as BxPC-3-mock. The retrovirus infection experiment was carried out twice, using BxPC-3-mock and BxPC-3-SMAD4 to produce batch 1 and batch 2.

13)-2 Confirmation of CD147 and SMAD4 performance

BxPC-3 (ATCC, Cat.CRL-1687), BxPC-3-mock and BxPC-3-SMAD4 prepared in Example 13)-1 were performed using Simple Western assays (Protein Simple Japan Co., Ltd., Wes). parse. As a SMAD4 positive control sample, MIA PaCa-2 was used. For detection of SMAD4, anti-SMAD4 antibody (R&D systems, Cat. AF2097) was used. For the detection of GAPDH, anti-GAPDH antibody (Abfrontier, Cat. LF-MA0026) was used. For the detection of CD147, anti-CD147 antibody (Abcam, Cat. Ab108317) was used.

The results are shown in Figure 25(a). MIA PaCa-2 is SMAD4 positive and CD147 positive. BxPC-3 is SMAD4 negative and CD147 positive. No effects caused by retroviral infection were observed in BxPC-3-mock, which was SMAD4 negative and CD147 positive. BxPC-3-SMAD4, which was introduced into SMAD4-expressing retroviral vector, was positive for SMAD4 in both batch 1 and batch 2. The SMAD4 performance of batch 2 was high, and in subsequent experiments, batch 2 was used as BxPC-3-SMAD4.

13)-3 Susceptibility of SMAD4 stable BxPC-3 tumors to the CD147 human chimeric antibody

2.5×10 ⁶ cells of BxPC-3-mock or BxPC-3-SMAD4 were suspended in PBS containing 100% Matrigel (Corning Company, Cat. 354234), and transplanted into BALB/c-nu mice of 5-week-old females. subcutaneously in the axillary part of mice (CAnN.Cg-Foxn1nu/CrlCrlj, purchased from Charles River, Japan). Based on tumor volume, groups were implemented 6 days after BxPC-3-mock transplantation and BxPC-3-SMAD4 3 days after transplantation. On the day of group implementation, 7, 14, 21, and 28 days later, 10 mg/kg was administered. Human chimeric anti-human CD147 antibody LN22R8R8chIgG2, or LN22R8R8chIgG4P was injected into the abdominal cavity of cancer-bearing mice (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 25(b) and Figure 25(c). In the figure, the mean value and standard error are combined for changes in tumor volume. BxPC-3-mock tumor lines showed resistance or hyposensitivity to LN22R8R8chIgG2 or LN22R8R8chIgG4P antibodies. The BxPC-3-SMAD4 tumor line showed partial susceptibility to LN22R8R8chIgG2 or LN22R8R8chIgG4P antibodies.

13)-4 Changes in p38 signaling in tumors caused by SMAD4 expression

It has been reported that in SMAD4-negative pancreatic cancer cells, p38 signal is enhanced by expressing SMAD4 (Chen et al., B.M.C., 1471-2407, 2014). Regarding changes in p38MAPK and phosphorylated p38MAPK in BxPC-3-SMAD4 tumors due to anti-human CD147 human chimeric antibody administration (72 hours after antibody administration), Simple was used in the same manner as in Example 13)-1. Analyzed by Western assays (Protein Simple Japan Co., Ltd., Wes). In the same manner as in Examples 1)-18, LN22R8chIgG4P was administered as an anti-human CD147 human chimeric antibody at 10 mg/kg to mice with MIA PaCa-2 subcutaneous tumors. For sample preparation from tumor tissue (n=3), gentleMACS Octo Dissociator with Heaters (Miltenyi Company) was used.

In FIG. 26(a) , the p38 MAPK measured values are shown in the figure by combining the average and standard error of the measured values of three tumor samples. In Figure 26(b), the measured values of phosphorylated p38 MAPK are combined and the average and standard error of the measured values of three tumor samples are combined and recorded in the figure.

In BxPC-3-SMAD4 tumors, p38 and phosphorylated p38 increased more than 2-fold. A decrease in the p38 fraction and an increase in the phosphorylated p38 fraction were observed 72 hours after administration of the CD147 human chimeric antibody.

In pancreatic cancer cell BxPC-3 tumors, it was found that the expression of SMAD4 leads to the enhancement of p38 signal. It is believed that the enhancement of SMAD4-dependent p38 signaling may contribute to the increased sensitivity of the CD147 human chimeric antibody LN22R8chIgG4P.

(Example 14) Anti-tumor effect of chimeric CD147 antibody in gemcitabine-resistant pancreatic cancer

To investigate the anti-tumor effect of CD147 antibody in gemcitabine-resistant pancreatic cancer tumor model. 5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 5-week-old female NOD-scid mice (NOD. CB17-Prkdc<scid>/J, Charles River, Japan) subcutaneously in the axillary area. Six days after transplantation, gemcitabine (Eli Lilly Co., Ltd., Japan), a therapeutic drug for pancreatic cancer, was administered intraperitoneally at 400 mg/kg. One week later, mice with confirmed gemcitabine-resistant tumors were Tumor size was divided into groups, and gemcitabine was intraperitoneally administered at 400 mg/kg on the 13th and 20th days after transplantation in the control group (n=5). As for the CD147 antibody and gemcitabine combined administration group (n=5), in addition to intraperitoneal administration of gemcitabine at 400 mg/kg on the 13th day after transplantation (group day) and 20th day, the group was administered intraperitoneally on the 13th day after transplantation (group day). ), and on day 20, the CD147 human chimeric antibody LN22R8chIgG4P was administered intraperitoneally at 10 mg/kg. The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

The results are shown in Figure 27. In the figure, the mean value and the standard error are combined with respect to the changes in tumor volume. The tumor proliferation in the non-treatment group (n=5) that was not administered gemcitabine and antibody was combined and recorded.

The average tumor size in the group administered gemcitabine as a control drug was 1269 mm ³ 28 days after transplantation. Compared to the group in which no shrinkage of tumors was observed, the average tumor size in the group administered the CD147 human chimeric antibody LN22R8chIgG4P was 15 mm ³ . Tumor regression was observed in 3 out of 5 cases. The results suggest that proliferating pancreatic cancer tumors that are resistant to gemcitabine may also be susceptible to CD147 antibodies.

(Example 15) Anti-tumor effect of humanized CD147 antibody in liver cancer model 15)-1 Expression of CD147 and SMAD4 in Hep G2 cells

Similar to Example 13)-2, the expression of CD147 and SMAD4 in the liver cancer cell line HepG2 cells (ATCC, Cat. HB-8065) was confirmed. As CD147-positive control specimens, MIA PaCa-2 and BxPC-3 (ATCC, Cat. CRL-1687) were used. MIA PaCa-2 was used as a SMAD4-positive control specimen, and BxPC-3 was used as a SMAD4-negative control specimen.

For the detected signal, the ratio of the signal to GAPDH is obtained and summarized below. It was found that Hep G2 cells were positive for CD147 and SMAD4.

CD147/GAPDH signal ratio

SMAD4/GAPDH signal ratio

15)-2 Confirmation of CD147 expression in Hep G2 cells using flow cytometry

CD147 expressed on the cell surface of HepG2 cells (ATCC, Cat.HB-8065) was analyzed using flow cytometry. To confirm the expression of human CD147, the APC-labeled anti-human CD147 mouse IgG1 antibody MEM-M6/1-APC (Thermofisher, Cat. MA1-10104) was used as a commercially available anti-human CD147 antibody. As a mouse IgG1 isotype control antibody, mIgG1-APC (Miltenyi Bio, Cat. 130-092-214) was used. To the HepG2 cell suspension, add 1/10 amount of MEM-M6/1-APC and treat at 4°C for 30 minutes. After washing the cells with PBS buffer containing 5% FBS, the cells were measured with a flow cytometer (CantoII, BD Bioscience), and the results were summarized in Figure 28(a) .

It was found that Hep G2 cells treated with MEM-M6/1-APC showed strong fluorescent signals expressing CD147.

15)-3 p38 activation by humanized CD147 antibody in hepatoma cells

In order to investigate the effect of anti-CD147 antibody on p38MAPK in liver cancer cell HepG2, 10 μg/ml anti-human CD147 human chimeric antibody (LN22R8chIgG4P) or the anti-human CD147 humanized antibody (# prepared in Example 6)-4-2 was used. HepG2 cells (ATCC, Cat.HB-8065) were treated with 84H1L2hIgG2, #84H1L2hIg4P, #101H1L2hIgG2, #101H1L2hIgG4P, #110H1L4hIg4P, #131H2L2hIgG2) for 15 minutes, using Simple Western assays (Protein Simple Japan Co., Ltd. , Wes) evaluated P38 phosphate change. As a control sample, HepG2 cells were similarly treated with 10 μg/ml of human IgG (hIgG, Jackson ImmunoResearch Co., Ltd. 009-000-003) and subjected to analysis. For the detection of p38MAPK, p38 MAPK rabbit mAb (Cell signaling technology company, Cat.#9212) was used. For the detection of phosphorylated p38MAPK, P-p38 MAPK(T180/Y182)(D3F9)XP rabbit mAb (Cell signaling technology company, #4511S) was used. The ratio of the detected phosphorylated p38MAPK signal to the p38MAPK signal is shown in Figure 28(b).

By treatment with LN22R8chIgG4P, #84H1L2hIgG2, #84H1L2hIg4P, #101H1L2hIgG2, #101H1L2hIgG4P, #110H1L4hIg4P or #131H2L2hIgG2, more than 2 times the phosphorylated p38 MAPK signal was observed in the human IgG treated group. The increase in number was found to be related to liver cancer cells Pancreatic cancer cells also induce phosphorylation of p38MAPK by anti-human CD147 antibodies.

15)-4 Comparison of the anti-tumor effects of humanized CD147 antibody and sorafenib in liver cancer

For the human liver strain HepG2 (ATCC, Cat. HB-8065), which is positive for CD147 and SMAD4 and has confirmed phosphorylation of p38 by anti-human CD147 antibodies, the anti-tumor effects of anti-human CD147 human chimeric antibodies and humanized antibodies were examined Effect.

5×10 ⁶ cells of the human liver strain HepG2 were suspended in PBS containing 50% Matrigel (Corning Company, Cat. 354234), and transplanted into 4-week-old female NOD-scid mice (NOD.CB17-Prkdc<scid> /J, purchased from Charles River, Japan, under the skin of the armpit. Based on the tumor volume, groups were implemented 9 days after transplantation, and the human chimeric CD147 antibody (LN22R8chIgG4P) and the humanized CD147 antibody (#84H1L2hIgG2, # prepared in Example 6)-4-2 were administered at 1 mg/kg and 10 mg/kg. 84H1L2hIg4P, #110H1L4hIg4P) were administered into the abdominal cavity of cancer-bearing mice (n=5). Sorafenib (Nexavar tablets 200 mg, Bayern Pharmaceutical Co., Ltd.) as a control drug for the treatment of liver cancer was used according to the reference literature attached to the instructions for Nexavar tablets (Chang et.al., Cancer Chem. Thera. Pharm., 2007), dissolved in PEG-35 castor oil (Cremophor EL, Nakarai Tesque Company, Cat. 09727-14) ethanol solvent, 30 mg/ kg, 90mg/kg were administered orally to cancer-bearing mice (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (manufactured by Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Table 7 and Figures 29(a) to (d). In the figure, the mean value and standard error are combined for changes in tumor volume.

Partial inhibition of tumor proliferation was observed with sorafenib administration, but tumor disappearance was not observed. All of the LN22R8chIgG4P, #84H1L2hIgG2, #84H1L2hIg4P, and #110H1L4hIg4P lines showed excellent anti-tumor effects accompanied by tumor disappearance.

(Example 16) Effect on T cells and PBMC

It has been reported that CD147 is accompanied by the activation of T cells and increases in CD4-positive and CD8-positive T cells (Hu et al., J. Cell. Mol Med., 2132-2143, 2010). In some CD147 antibodies, It has the effect of inhibiting the activation induction ability and proliferation of T cells (Koch et al., Int. Immunology, 777-786, 1999; Chiampanichayakul et al., Immunology 167-178, 2006). The effect of an anti-human CD147 antibody that shows strong anti-tumor effect independently of effector function on peripheral blood lymphocytes (PBL) containing T cells was investigated.

16)-1 Increased expression of CD147 caused by CD3/CD28 stimulation of PBMC

Human PBMC were used to investigate whether CD147 expression increases with T cell activation. Human PBMC were cultured in PMI1640 medium containing 10% FBS at 37°C in the presence of 5% _CO2 . Dynabeads Human T-Activator CD3/CD28 (CD3/CD28 beads, Thermofishers scientific, Cat. 1131D) was added during culture to induce proliferation, and flow cytometric analysis was performed 4 days later to investigate whether the expression of CD147 changed. To confirm the expression of human CD147, the commercially available APC-labeled anti-human CD147 mouse IgG1 antibody MEM-M6/1-APC (CD147-APC, Thermofisher, Cat. MA1-10104) as an anti-human CD147 antibody was used. mIgG1-APC (Miltenyi Bio, Cat. 130-092-214) was used as a mouse IgG1 isotype control antibody. In order to detect CD3, CD4, and CD8 in T cells contained in human PBMC, APC/Fire ^TM 750 anti-human CD3 antibody (manufactured by BioLegend, Cat. 344840) and PerCP/Cy5.5 anti-human CD4 (manufactured by BioLegend, Cat. .344608), Brilliant Violet 510 anti-human CD8 (BioLegend, Cat. 344732).

The binding of CD147-APC in CD3 and CD4 positive cells and CD3 and CD8 positive cells is summarized in Figure 30. Regarding CD3 and CD4-positive cells and CD3 and CD8-positive cells, it was confirmed that the expression of CD147 increased when stimulated by CD3/CD28 beads, and it was confirmed that the expression of CD147 on the T cell membrane surface increased with the activation of T cells.

16)-2 Evaluation of the role of anti-human CD147 antibodies in the proliferation of human peripheral blood mononuclear spheres

Analysis of the role of anti-human CD147 antibodies in the proliferation of human peripheral blood mononuclear cells (PBMC). 2P10F2chIgG4P was used as anti-human CD147 antibody. Using CellVue Claret Far Red Fluorescent Cell Linker Kit (sigma, Cat.MIDCLARET-1KT), PBMC were fluorescently labeled, and then cultured in RPMI1640 medium containing 10% FBS at 37°C in the presence of 5% _CO2 . During culture, IL-2 and Dynabeads Human T-Activator CD3/CD28 (CD3/CD28 beads, Thermofishers scientific, Cat. 1131D) were added. When inducing proliferation, 2P10F2chIgG4P (10 μg/ml) was added to investigate the effect on proliferation. On the 3rd and 5th days of culture, the fluorescence signal of PBMC cells decreased due to cell division was measured using a flow cytometer (CantoII, BD Bioscience), and the results are summarized in Figure 31 .

By adding CD3/CD28 beads, reduced PBMC cell fluorescence signals due to cell division were observed after 3 days and 5 days of culture. When IL-2, 2P10F2chIgG4P, or IL-2 and 2P10F2chIgG4P were added during culture, no change was seen in the decrease in fluorescent signal. It is suggested that the anti-human CD147 human chimeric antibody 2P10F2chIgG4P, which showed strong anti-tumor effect in the mouse tumor model of human pancreatic cancer shown in Examples 1) to 18, did not affect the proliferation of PBMC.

16)-3 Evaluation of anti-human CD147 antibodies in interleukin production by human peripheral blood lymphocytes

Human chimeric antibodies #84chIgG1, #84chIgG2, #84chIgG4P, #84chIgG1LALA, #84chIgG4PFALA, #101chIgG4P or #110chIgG4P were used as anti-human CD147 antibodies. PBL was investigated using Ficoll-Paque PLUS (GE HEALTHCARE Japan Co., Ltd.) derived from human peripheral blood. Spread 10 μg/ml human chimeric antibody in a 96-well plate. Human IgG (hIgG, Jackson ImmunoResearch, 009-000-003) was used as a negative control antibody, and Dynabeads Human T-Activator CD3/CD28 (CD3/CD28 beads, Thermofishers scientific, Cat. 1131D) was used to induce T cell activity. or cytokine-induced positive control antibodies. Add 1x10 ⁶ PBL to the antibody-coated well, and 24 hours later, measure the interleukins (IL2, TNFα, INFγ) in the culture medium. Dynabeads Human T-Activator CD3/CD28 was added directly to the well where PBL was added. Similarly, the interleukins (IL2, TNFα, INFγ) in the culture medium were measured for 24 hours. For the measurement of IL2, Quantikine ELISA Human IL-2 (R&D systems, Cat.D2050) was used. For the measurement of TNFα, Amersham TNF-αHuman, Biotrak Easy ELISA (GE HEALTHCARE Japan Co., Ltd., Cat. RPN5967) was used. For the measurement of INFγ, Human IFN-γ ELISA development kit (MABTECH, Cat. 3420-1H-6) was used. The measurement system was carried out in three groups, and the average value and standard deviation of the detected absorbances were calculated and summarized in Figure 32.

Any of the measured interleukins (IL2, TNFα, INFγ) produced by PBL was confirmed to increase by culturing in the wells where only CD3/CD28-beads were added as a positive control, and any one of them was coated with anti-human CD147 human embedded The same increase in interleukins as the negative control hIgG was not observed in the culture in the antibody-containing wells.

(Example 17) X-ray crystal structure analysis of humanized #110Fab’-CD147 complex 17) Crystallization of the-1 complex

Fab’2 obtained by cleaving humanized #110H1L4hIgG4P with pepsin was reduced with dithiothreitol and alkylated with iodoacetamide to obtain the Fab’ fragment. The mixture of this Fab' fragment and hCD147v2 (22-205) used in Example 5) was subjected to filtration chromatography using a Superdex 10/300GL Increase column (GE Healthcare) to obtain a complex fraction. The complex system was concentrated to 13g/L by replacing the buffer (10mM Tris HCl pH 7.5, 50mM NaCl) with AmiconUltra15 MWCO 10K (Millipore). The complex solution is crystallized by vapor diffusion method. Add a precipitant solution (0.1M NaMalonate pH 7.0, 12% (w/v) polyethylene glycol 3350) in an equal amount to 0.5 μL of the protein solution, and place it in a closed container with 0.05 mL of the precipitant solution. The two solutions were allowed to stand at 25°C without contacting each other. The 0.15 mm × 0.15 mm × 0.3 mm crystal obtained after about one week was immersed in a 30% (w/v) precipitant solution to which polyethylene glycol 400 was added, and then frozen in liquid nitrogen. X-ray diffraction data were collected using the beam PF-BL17A of the Synchrotron Facility Photon Factory (Ibaraki Prefecture, Japan). From the obtained diffraction image, the diffraction intensity was digitized using the software mosflm (CCP4: Collaborative Computational Project No. 4), and the crystal structure factor was obtained. The crystal space group P21, the crystal unit grid system (a=64.96Å, b=93.37Å, c=98.31Å, alpha=gamma=90, beta=90.89).

17)-2 Structural analysis of complex

Using the obtained homology model of the structural factors and Fab' fragments and the three-dimensional structural coordinates of the known structure of human CD147 (PDBID: 3b5h), molecular substitution method was carried out and the phase was determined. The calculation uses the software phaser (CCP4: Collaborative Computational Project No.4). The crystal system contains two complexes in asymmetric units. The software Refmac5 (CCP4: Collaborative Computational Project No. 4) is used to refine the structure, and the software coot is used to modify the model. Repeating this operation, the final R value of 23% and the free R value of 28% can be obtained with 2.3Å decomposition. The final model contained two Fab’ fragments of humanized #110H1L4 and hCD147v2 bound to each. Furthermore, in one case of hCD147v2, electron density corresponding to amino acid residues 23-203 was observed, but in the other case, the electron density corresponding to domain 1 was not clear, and only the electron density corresponding to amine was observed. Electron density of amino acid residues 103-202.

17)-3 Specification of epitope

The amino acid residues of hCD147v2 contained in the two complexes of the asymmetric unit and within 4 Å of the binding interface of the Fab' fragment of humanized #110H1L4 are as follows: Arg106, Lys108, Ala109, Val110, Lys127, Ser128 , Glu129, Ser130, Val131, Pro132, Pro133, Val134, Gln164, Gly165. A ribbon model and surface of the entire complex are presented in Figure 41, and the interaction of hCD147v2 and humanized #110H1L4 is presented in Figure 42.

(Example 18) Anti-tumor effect of humanized CD147 antibody in gastric cancer model

The anti-tumor effects of anti-human CD147 human chimeric antibodies and humanized antibodies were examined on human gastric cancer cell line KATO III cells (ATCC, Cat. HTB-103) confirmed to be CD147 positive by flow cytometry.

5×10 ⁶ cells of the human gastric cancer line KATO III were suspended in 100% Matrigel (Corning Company, Cat. 354234) and transplanted into 5-week-old female NOD-scid mice (NOD.CB17-Prkdc<scid>/J , purchased from CLEA (Japan) under the skin of the armpit. Grouping was performed based on tumor volume 3 days after transplantation. The human chimeric CD147 antibody (LN22R8chIgG4P) and the humanized CD147 antibody (#110H1L4hIg4P) prepared in Example 6)-4-2 were divided into groups at 10 mg/kg for 7 days each. Administered into the abdominal cavity of cancer-bearing mice (n=6). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 43. In the figure, the mean value and standard error are combined for changes in tumor volume.

The average tumor volume of mice in the untreated group was 290 mm ³ 24 days after transplantation, the LN22R8chIgG4p-administered group was 199 mm ³ , and the #110H1L4hIgG4P-administered group was 134 mm ³ . The calculated anti-tumor effect was based on the LN22R8chIgG4p-administered group. 31%, and 54% in the #110H1L4hIgG4P-administered group.

(Example 19) Anti-tumor effect of humanized CD147 antibody in chronic myelogenous leukemia model

The anti-tumor effect of anti-human CD147 humanized antibodies was examined on the human chronic myelogenous leukemia cell line KU812 cells (ATCC, Cat. CRL-2099) confirmed to be CD147 positive by flow cytometry.

5×10 ⁶ cells of the chronic myelogenous leukemia cell line KU812 were suspended in PBS containing 50% Matrigel (Corning Company, Cat. 354234), and transplanted into 5-week-old female NOD-scid mice (NOD.CB17-Prkdc <scid>/J, purchased from CLEA, Japan, under the skin of the armpit. Grouping was performed based on tumor volume 3 days after transplantation. The humanized CD147 antibody (#110H1L4hIg4P) prepared in Example 6)-4-2 was divided into groups at 10 mg/kg and administered to the abdominal cavity of cancer-bearing mice on 7 days each. Within (n=5). Imatinib (ASTATECH, Cat.#63168), a therapeutic drug for chronic myelogenous leukemia as a control drug, was prepared into a 9 mg/ml solution with distilled water, and 90 mg/kg was orally administered to cancer-bearing mice (Saturday). And stop taking the medicine on Sunday and give it continuously, and give it 4, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 21, 22, 23, and 24 days after transplantation). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 44. In the figure, the mean value and standard error are combined for changes in tumor volume.

Compared with the average tumor volume of mice in the untreated group, which was 627 mm ³ 25 days after transplantation, the imatinib-administered group was 328 mm ³ , and the #110H1L4hIgG4P-administered group was 6 mm ³ . The calculated anti-tumor effect was based on imatinib administration. The rate in the group administered with #110H1L4hIgG4P was 48%, and the rate in the group administered #110H1L4hIgG4P was 97%. Complete tumor regression was observed in 4 out of 5 cases only in the #110H1L4hIgG4P-administered group.

(Example 20) Anti-tumor effect of humanized CD147 antibody in colorectal cancer model

The anti-tumor effect of humanized antibodies was examined on human colorectal cancer cell line SW620 cells (ATCC, Cat. CCL-227), which was confirmed to be CD147 positive by flow cytometry.

5×10 ⁶ cells of human colorectal cancer line SW620 were suspended in 100% Matrigel (Corning Company, Cat. 354234) and transplanted into 5-week-old female NOD-scid mice (NOD.CB17-Prkdc<scid>/J , purchased from CLEA (Japan) under the skin of the armpit. Grouping was performed based on tumor volume 3 days after transplantation. The human chimeric CD147 antibody (LN22R8chIgG4P) and the humanized CD147 antibody (#084H1L2hIg4P or #110H1L4hIg4P) prepared in Example 6)-4-2 were divided into groups and then administered at 10 mg every 7 days. /kg was administered into the abdominal cavity of cancer-bearing mice (n=5). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 45. In the figure, the mean value and standard error are combined for changes in tumor volume.

The average tumor volume of mice in the untreated group was 1302 mm ³ 21 days after transplantation, the #084H1L2hIg4P-administered group was 709 mm ³ , and the #110H1L4hIgG4P-administered group was 403 mm ³ , and the calculated anti-tumor effect was based on #084H1L2hIg4P administration. The rate was 46% in the #110H1L4hIgG4P-administered group, and 69% in the #110H1L4hIgG4P-administered group.

(Example 21) Anti-tumor effect of humanized CD147 antibody in renal cancer 786-O model

The anti-tumor effect of humanized antibodies was examined in human renal carcinoma 786-O, which was confirmed to be CD147 positive by flow cytometry.

5×10 ⁶ cells of human renal carcinoma 786-O were suspended in 50% Matrigel (Corning Company, Cat. 354234) and transplanted into 5-week-old female NOD-scid mice (NOD.CB17-Prkdc<scid>/ J. Subcutaneously applied to the armpit area purchased from CLEA, Japan. Grouping was performed based on tumor volume 3 days after transplantation. The human chimeric CD147 antibody (LN22R8chIgG4P) and the humanized CD147 antibody (#084H1L2hIg4P or #110H1L4hIg4P) prepared in Example 6)-4-2 were divided into groups every 7 days after transplantation. 10 mg/kg was administered into the abdominal cavity of cancer-bearing mice for a total of 4 times (n=6). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 46. In the figure, the mean value and standard error are combined for changes in tumor volume.

The average tumor volume of mice in the untreated group was 918 mm ³ 31 days after transplantation, compared to 224 mm ³ in the #084H1L2hIg4P-administered group, and 379 mm ³ in the #110H1L4hIgG4P-administered group. The calculated anti-tumor effect was based on #084H1L2hIg4P administration. The rate in the group administered with #110H1L4hIgG4P was 76%, and the rate in the group administered #110H1L4hIgG4P was 59%.

(Example 22) Anti-tumor effect of humanized CD147 antibody in acute myeloid leukemia (AML) model

The anti-tumor effect of humanized antibodies was examined on the human AML cell line OCI-AML3 cells (DSMZ, Cat. ACC 582), which was confirmed to be CD147 positive by flow cytometry.

5×10 ⁶ cells of the human AML cell line OCI-AML3 were suspended in 50% GFR-Matrigel (Corning Company, Cat. 354230) and transplanted into 5-week-old female NOD-scid mice (NOD.CB17-Prkdc <scid>/J, purchased from CLEA, Japan, under the skin of the armpit. Grouping was performed based on tumor volume 3 days after transplantation, and 10 mg/kg of humanized CD147 produced in Example 6)-4-2 was administered from the tail vein every 7 days after the cancer-bearing mice were grouped (n=6). Antibody (#110H1L4hIg4P). The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

Tumor volume (mm ³ ) = 1/2 × short diameter (mm) × short diameter (mm) × long diameter (mm)

The results are shown in Figure 47. In the figure, the mean value and standard error are combined for changes in tumor volume.

The average tumor volume of mice in the untreated group was 1533 mm ³ 21 days after transplantation, and that of the #110H1L4hIgG4P-administered group was 394 mm ³ . The calculated anti-tumor effect was 74% in the #110H1L4hIgG4P-administered group.

(Example 23) Activity measurement of humanized anti-CD147 antibodies with different binding properties

5 × 10 ⁶ cells of the human pancreas strain MIA PaCa-2 were suspended in PBS containing 50% GFR-Matrigel (Corning Company, Cat. 354230), and transplanted into 4-week-old female Nude mice (BALB/c Slc -nu/nu, purchased from Japan SLC (Co., Ltd.) under the skin of the armpit. Grouping was performed based on tumor volume, and 10 mg/kg of the humanized CD147 antibodies (#110H1L4hIgG4P, # 110H13L02hIgG4P and #110H13L12hIgG4P, binding properties are reported in Table 6) to cancer-bearing mice (n=6). On days 3 and 10 after transplantation, gemcitabine (purchased from Eli Lilly, Japan), which is a control drug and is a therapeutic drug for pancreatic cancer, was administered to cancer-bearing mice (n=6) at 400 mg/kg through the tail vein. The long diameter and short diameter of the transplanted tumor were measured twice a week using an electronic digital caliper (Mitutoyo Co., Ltd.), and the tumor volume was calculated using the calculation formula shown below.

The results are shown in Figure 48. In the figure, the mean value and standard error are combined for changes in tumor volume.

Compared to the control drug gemcitabine, the tumor growth inhibition rate was 66%, and any humanized CD147 antibody showed a better anti-tumor effect than gemcitabine in the 10 mg/kg administration group.

(Example 24) Comparison of CD147 antibodies 24)-1 Evaluation of antigen binding properties of existing anti-CD147 antibodies

As competitive antibodies, purified antibodies were prepared based on the patented sequences of 4A5 and 5F6 antibodies in WO2010/036460 and PPAT-082-03 in WO2017/061602. To measure the dissociation constant with CD147 protein, Biacore T200 (manufactured by GE Healthcare Bioscience) was used, and Anti-Human IgG (Fc)antibody immobilized using Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience) was used, using the antibody as the ligand. Capture is a capture method in which an antigen is measured as an analyte. As the running buffer, HBS-EP+ (manufactured by GE Healthcare Bioscience) was used, and as the sensor wafer, CM5 (manufactured by GE Healthcare Bioscience) was used. After adding 1 μg/mL competitive antibody to the chip at 10 μL/min for 60 seconds, add the dilution series solution of the antigen used in Example 2)-5 (0.5~8 μg/mL) at a flow rate of 30 μL/min for 120 seconds, and then, Detect dissociation phase for 300 seconds. As a regeneration solution, 3M magnesium chloride (manufactured by GE Healthcare Bioscience) was added at a flow rate of 20 μL/min for 30 seconds. In the analysis of data, a 1:1 binding model was used to calculate the association rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD; KD=kd/ka). The dissociation constant of the humanized CD147 antibody (#084H1L2hIgG4P or #110H1L4hIgG4P) was calculated according to the method of Example 7)-1.

Table 8 shows the calculated dissociation constants, effector functions, and information on epitope regions.

24)-2 Competitive ELISA with existing CD147 antibodies

The humanized CD147 antibody (#084H1L2hlg4P or #110H1L4hIg4P) prepared in Example 6)-4-2 was used to evaluate the binding property to recombinant human CD147/Fc (Sino Biological, Cat. 10186-H02H) by competition ELISA. As competitive antibodies, 4A5, 5F6 antibodies and PPAT-082-03 antibodies prepared with 24)-1 were used. As a competition negative control antibody, human IgG (JACKSON, Cat. 130093) was used. As competition positive control antibodies, for #084H1L2hIg4P antibody, use #84H1L2hIgG2 antibody, and for #110H1L4hIg4P, use #110chIgG2 antibody.

Recombinant human CD147/Fc diluted in PBS was added to a 96-well plate (Thermo Scientific, Cat. 43454) at 2ug/ml, 50ul/well, and stored at 4°C overnight. After removing the protein solution, add 300ul of PBS containing 1% BSA and warm at room temperature for 1 hour. Remove the solution, add 25u of existing antibody solution (0, 0.2, 2, 20ug/ml), and heat at room temperature for 2 hours. Add 25ul humanized CD147 antibody (#084H1L2hIg4P or #110H1L4hIg4P) to each well at a concentration of 20ng/ml and incubate at room temperature for 2 hours. After washing twice with PBS containing 0.05% Tween20 (BIO RAD, Cat. 170-6531), 50 ul of anti-human IgG4-HRP (Abcam, Cat. ab99823), incubate at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween20 (BIO RAD, Cat. 170-6531), the washing fluid was fully removed, and 50 ul of HRP matrix solution (eBioscience, Cat. 00-4203-58) was added. After heating at room temperature for 15 to 20 minutes, the absorbance at 405 nm was measured with a flat disk reader (PerkinElmer Company, model name: EnVision2104).

The results are shown in Table 8 and Figures 49(a) and (b).

The binding of #084H1L2hlg4P to recombinant human CD147/Fc is inhibited in the presence of hIgG in the presence of existing CD147 antibodies at 1~10ug/ml. The binding of #110H1L4hIg4P to recombinant human CD147/Fc was not inhibited in the presence of hIgG and existing CD147 antibodies at 0.1~10ug/ml, and was inhibited in the presence of #110chIgG2 antibody. It was found that although the epitope of #084H1L2hlg4P is subject to competition due to the binding of existing anti-CD147 antibodies, the epitope of #110H1L4hlg4P is not affected by the binding of existing anti-CD147 antibodies.

The epitope information of the H110H1L4h antibody shown in Example 17)-3 is shown in Table 8. Based on the binding competition test results (Example 2)-8) with the 2P10F2 antibody in which the epitope was presumed based on the analysis of the CD147 variant in Example 1)-9, the epitope region of #084H1L2h was deduced and described. in Table 8.

24)-3 ADCC evaluation of anti-CD147 antibodies

The ADCC activity of the anti-CD147 antibody was evaluated according to the methods of Examples 1)-15. As different conditions from the method of Examples 1)-15, HepG2 cells (ATCC, Cat. HB-8065) were used as ADCC target cells, and #110H1L4hIgG4P, #084H1L2hIg4P, 4A5, 5F6, and PPAT-082-03 were used as CD147 antibodies. , evaluated at a concentration of 1 μg/ml. The measurement system was carried out in three groups, and the average value and standard deviation were calculated. When ⁵¹ Cr is detected in more than 5% of the cells, it is regarded as ADCC positive (+). The case of less than 5% was regarded as negative ADCC activity (-). The results are shown in Table 8.

No ADCC activity was detected in the #110H1L4hIgG4P and #084H1L2hIg4P lines, and they were judged to be ADCC(-). 4A5, 5F6, and PPAT-082-03 lines had ⁵¹ Cr detected in more than 5% of the cells and were set as ADCC positive (+). #110H1L4hIgG4P and #084H1L2hIg4P are ADCC negative. Although they have the possibility of inducing cell death in normal human cells such as blood cells expressing CD147 in vivo, they are predicted to be lower than those of 4A5, 5F6 and PPAT-082-03 which are ADCC active positive. .

24)-4 CDC evaluation of anti-CD147 antibodies

The complement-dependent cytocidal activity (CDC activity) caused by the anti-human CD147 antibody was evaluated according to the procedures of Examples 1) to 16. As different conditions from the methods of Examples 1)-16, human liver strain HepG2 cells (ATCC, Cat. HB-8065) were used as target cells, and #110H1L4hIgG4P, #084H1L2hlg4P, 4A5, 5F6 and PPAT-082-03 were used as target cells. Anti-human CD147 antibody was measured by adding rabbit complement to a final concentration of 8%. The measurement system was carried out in three groups, and the average value and standard deviation were calculated. Antibodies in which antibody-dependent CDC activity was observed in 30% or more were regarded as positive for CDC activity and were recorded as CDC(+) in the table. The results are shown in Table 8. Only 4A5 of the anti-human CD147 antibody system showed positive CDC activity. #110H1L4hIgG4P, #084H1L2hIg4P, 5F6, and PPAT-082-03 are CDC negative. Although there is a possibility of inducing cell death in normal human cells such as blood cells expressing CD147 in vivo, it is predicted to be lower than that of 4A5, which is positive for CDC activity. .

24)-5 ADCP evaluation of anti-CD147 antibodies

According to the method of Examples 1)-17, the ADCP activity of the anti-CD147 antibody was measured. As different conditions from the method of Examples 1)-17, human liver strain HepG2 cells (ATCC, Cat. HB-8065) were used as target cells, and #110H1L4hIgG4P, #084H1L2hlg4P, 4A5, 5F6, and PPAT-082-03 were used as target cells. Anti-CD147 antibody was added at a concentration of 1 μg/ml, and equal amounts of ADCP-labeled cells were added to the labeled RAW264.7 cell line to measure ADCP activity. The measurement system was carried out in 3 groups, and the average value and standard deviation were calculated. An increase in ADCP activity of less than 10% compared with the human IgG-treated group was regarded as weak positive (±), and an increase in activity of more than 10% was regarded as positive (+). Shown in Table 8.

The ADCP activity of #110H1L4hIgG4P is less than 10%, and it is judged as ADCC activity ±. The ADCP activity of #084H1L2hIgG4P, 4A5, 5F6, and PPAT-082-03 antibodies is more than 10%, and it is judged as ADCP activity +. The #110H1L4hIgG4P line, which recognizes CD147-D2, has lower ADCP activity than other CD147 antibodies that recognize CD147-D1. After ADCP, CD147 antibody binds to CD147, through the Fcγ receptor expressed on macrophages or monocytes, although it is necessary to recognize the FC part of the antibody, the epitope of #110 antibody is close to the cell surface and is an antigen. The FC part of the CD147-binding antibody is more difficult to recognize Fcγ receptors than other CD147-D1 antibodies, and there is a possibility that the ADCP activity is low. #110H1L4hIgG4P and ADCP activity are weakly positive. Although there is a possibility of inducing cell death in normal human cells such as blood cells expressing CD147 in vivo, #084H1L2hIg4P, 4A5, 5F6, and PPAT-082 are predicted to be more positive than ADCP activity. -03 lower.

(Example 25) 25)-1 Anti-CD147 antibody-induced agglutination of blood cells

Some anti-CD147 antibodies have been reported to induce agglutination of blood cells (Kasinrerk et al., Immunology 1999, 96(2) p184-192). Aggregation of blood cells may cause serious hematological toxicity (Doll, C., et al., 1994, Curr. Opin. Oncol., 345-350), which is an undesirable property for therapeutic antibodies. Differences in cell accumulation activity were investigated against anti-CD147 antibodies. As CD147 antibodies, #110H1L4hIgG4P, #084H1L2hlg4P, 4A5, 5F6, and PPAT-082-03 were evaluated. As a negative control antibody, human IgG (hIgG, ChromPure Human IgG, Jackson ImmunoResearch Laboratories, Cat. 009-000-003) was used. HEL92.1.7 cells (modified from ATCC, Cat.#TIB-180) were added to each well of a 96-well U chassis (Sumitomo Bakelite Co., Ltd., Cat.MS-9096U) at 1600 cells/80uL containing 10% FBS (HyClone Company, Cat. SH30084.03) RPMI1640 medium (Thermo Fisher Scientific Company, Cat. 11875-093), cultured for 4 hours under the conditions of 5% CO ₂ , humidity 95%, and 37 degrees. In each well, add 20ul of anti-CD147 antibody solution (150ug/ml, 50ug/ml) to make a final concentration of 30, 10ug/ml. Incubate for 2 days under conditions of 5% CO2, 95% humidity, and 37 degrees, and observe under a microscope.

Compared to the addition of human IgG, #110H1L4hIgG4P, cell accumulation was not observed. In the presence of #084H1L2hIg4P, 4A5, 5F6, and PPAT-082-03 antibodies, cell accumulation was observed, and a cell mass folded in the center of the plate was observed. The #110H1L4hIgG4P series that recognizes CD147-D2 is different from other CD147 antibodies that recognize the CD147-D1 domain and shows no agglutination activity on blood cells. CD147 antibodies, including #084, which have cell agglutination activity, may cause toxicity such as thrombosis through agglutination of blood cells when administered to humans. It is expected that the amount of heparin or low dosage used in the treatment of thrombosis can be Subcutaneous injection of molecular heparin or combined use of antiplatelet drugs can avoid or reduce side effects.

25)-2 Risk assessment of interleukin release syndrome

Through the administration of therapeutic antibodies, some antibodies such as OKT3 and TGN1412 activate immune cells and increase the level of interleukins in the blood, causing severe interleukin release syndrome (Gaston, R., Kidney International, 1991, 141-148; Suntharalingam, G., et al., N. Engl. J. Med. 2006, 1018-1028). It has been reported that some CD147 antibodies have an effect on immune cells and increase the production of interferon γ and interleukin-4 (Hu, J., et al., J. Cell. Mol. Med., 2010, 2132-2143 ). The toxicity of antibody drugs caused by this interleukin release syndrome can be predicted by interleukin release analysis using peripheral blood (Vessillier, S. et al., J. Immunolol. Methods, 2015, 43-52). The risk of interleukin release syndrome was assessed by the same human peripheral blood interleukin release assay. As CD147 antibodies, #110H1L4hIgG4P, #110chIgG4ProFALA, #084H1L2hIg4P, and #084H1L2hIg2 were used. As comparative antibodies, bevacizumab (Genentech, Inc.), trastuzumab (Roche Pharma AG), Alemtuzumab (Sanofi Co., Ltd.), anti-human CD3 antibody (BioLegend Cat. No317326). For all CD147 antibodies evaluated, no increase in cell proliferation or interleukin release (TNFα, INF-γ, IL-2, IL- 6. The impact of IL-8, IL-10, MIP-1α) is weaker than that of bevacizumab, which has a low risk of interleukin release syndrome. With anti-human CD3 antibody (OKT3), increased cell proliferation and increased release of interleukins (TNFα, INF-γ, IL-2, IL-6, IL-8, IL-10, MIP-1α) were observed . The #110H1L4hIgG4P, #110chIgG4ProFALA, #084H1L2hIg4P, and #084H1L2hIg2 lines showed no possibility of inducing interleukin release leading to interleukin release syndrome.

25)-3 Safety evaluation of anti-CD147 antibody in monkeys

It has been reported that some anti-mouse CD147 antibodies induce the accumulation of red blood cells in the spleen by inhibiting the function of CD147 when administered to mice, inhibiting the amount of red blood cells in peripheral blood and causing anemia (Coste, I. et al. ., Blood, 2001, 3984-3988). The CD147 antibodies such as #110H1L4hIgG4P obtained in the present invention have not been shown to bind to mouse CD147, so safety evaluation in mice is not appropriate. Therefore, #110H1L4hIgG4P, an anti-CD147 antibody whose binding properties to human and monkey CD147 were confirmed by experiments using flow cytometry, was administered to cynomolgus monkeys and its safety was evaluated. Results of a single intravenous administration of #110H1L4hIgG4P at the maximum possible dose of 99.2 mg/kg in cynomolgus monkeys (one male and one male) during the observation period up to 15 days after the administration and the pathology at the end of the observation period Histological examination showed no serious toxicity (changes in body weight, feed intake, and pathological changes). #110H1L4hIgG4P showed no toxicity to cynomolgus monkeys and showed potential for use in cancer treatment in humans.

(Example 26) Effect of KLF5 on CD147 antibody sensitivity

In cancer cells, Lethal EMT signal is known to be a SMAD2/SMAD3/SMAD4-dependent cell death signal. It has been reported that this Lethal EMT signal, in SMAD4-negative cancer cells, is a protein of the transcription factor KLF5 that is usually inhibited by SMAD signaling. Performance increases and lethality signals are suppressed (David, C. Cell, 2016, 1015-1030). The CD147 antibody of the present invention activates SMAD signals and exhibits anti-tumor effects on SMAD4-positive cells, thereby inducing SMAD signal-dependent cell death. To investigate whether KLF5 is involved in the susceptibility to CD147 antibody-dependent anti-tumor effects.

26)-1 Production of KLF5 expression strain

According to the method of Example 13, a stable expression strain of KLF5 in MIA PaCa-2 cells was produced. The amino acid sequence and nucleotide sequence of human KLF5 are shown in Sequence ID Nos. 145 and 146, respectively. As the KLF5 gene, a retroviral vector pQCXIP incorporating the sequence (Ref seq. ID: NM_001730.4) contained in (Genscript Co., Cat. OHu21278C) was prepared and used for producing retrovirus. The retrovirus was incorporated into the chromosome by virus infection, and puromycin-resistant, KLF5-positive MIA PaCa-2 cells were selected, resulting in KLF5-positive MIA PaCa-2 cells and MIA PaCa-2-KLF5. The retroviral vector pQCXIP was infected in the same manner, and puromycin-resistant MIA PaCa-2 cells were used as MIA PaCa-2-mock.

26)-2 Confirmation of CD147 and SMAD4 performance

Based on flow cytometry, the expression of CD147 in MIA PaCa-2-mock and MIA PaCa-2-KLF5 was confirmed. According to the method of Example 13-2, it was confirmed that the expression amount of KLF5 in MIA PaCa-2-KLF5 increased from MIA PaCa-2-mock. For the detection of KLF5, KLF5 antibody (CST Company, Cat.#51586) was used.

26)-3 Susceptibility of KLF5-expressing MIA PaCa-2 tumors to humanized CD147 antibodies

According to the method of Example 7)-2, the susceptibility of MIA PaCa-2-KLF5 and MIA PaCa-2-mock tumors to humanized CD147 antibody #110H1L4hIgG4P was compared. Three days after the cell transplantation, the humanized CD147 antibody (#110H1L4hIg4P) prepared in Example 6)-4-2 was administered to the tail vein of the cancer-bearing mice at 1 mg/kg (n=6). Antibodies were administered in the same manner 7 days later. In the control group of cancer-bearing mice, PBS buffer was similarly administered into the tail vein (n=6). The results are shown in Figure 50.

After 14 days of administration of humanized CD147 antibodies, the average tumor volume of MIA PaCa-2-mock tumors was reduced to 9% of the control group, indicating sensitivity to CD147 antibodies. The average tumor volume of MIA PaCa-2-KLF5 was 80% of that of the control group, indicating low sensitivity to CD147 antibodies. It is known that the SMAD signal-dependent anti-tumor effect caused by CD147 antibodies is inhibited by the expression of KLF5.

Industrial utilization possibilities

According to the present invention, there is provided a CD147-specific antibody that activates CD147 and exhibits high anti-tumor effect. According to the present invention, antibodies showing high anti-tumor effects independent of effector functions are provided. The antibody system of the present invention exhibits significantly stronger efficacy on liver cancer cells than sorafenib, which is one of the standard therapeutic drugs for liver cancer. The antibody system of the present invention shows a significantly stronger medicinal effect on pancreatic cancer cells than gemcitabine, which is one of the standard therapeutic drugs for pancreatic cancer. Furthermore, the antibody system of the present invention shows significantly stronger efficacy on chronic myelogenous leukemia cells than imatinib, which is one of the standard therapeutic drugs for chronic myelogenous leukemia. The antibody of the present invention provides an anti-CD147 antibody whose anti-tumor effect does not depend on effector function, and which is less problematic and has excellent safety in other safety evaluation items. Although CD147 is expressed not only in tumor cells but also in blood cells, the antibody of the present invention has no effect on T cells and PBMC and is not dependent on effector function. Therefore, it has so-called safety when developing as an anti-tumor agent. The advantage of having less worries about sex. According to the present invention, there are provided pharmaceutical compositions containing the above-mentioned antibodies, and methods for treating tumors using the antibodies and/or the pharmaceutical compositions.

[Sequence listing non-keyword text]

Sequence ID 1: Amino acid sequence of variant 1 of human CD147

SEQ ID NO: 2: Nucleotide sequence of variant 1 of human CD147

SEQ ID NO. 3: Amino acid sequence of variant 2 of human CD147

SEQ ID NO: 4: Nucleotide sequence of variant 2 of human CD147

SEQ ID NO: 5: Amino acid sequence of human SMAD4

SEQ ID NO: 6: Nucleotide sequence of human SMAD4

SEQ ID NO: 7: Nucleotide sequence of the variable region of the light chain of LN22R8

SEQ ID NO: 8: Amino acid sequence of the variable region of the light chain of LN22R8

SEQ ID NO: 9: Nucleotide sequence of the variable region of the heavy chain of LN22R8

SEQ ID NO. 10: Amino acid sequence of the variable region of the heavy chain of LN22R8

SEQ ID NO. 11: Amino acid sequence of CDRL1 of LN22R8

SEQ ID NO. 12: Amino acid sequence of CDRL2 of LN22R8

SEQ ID NO. 13: Amino acid sequence of CDRL3 of LN22R8

SEQ ID NO. 14: Amino acid sequence of CDRH1 of LN22R8

SEQ ID NO. 15: Amino acid sequence of CDRH2 of LN22R8

SEQ ID NO. 16: Amino acid sequence of CDRH3 of LN22R8

SEQ ID NO: 17: Nucleotide sequence of the variable region of the light chain of 2P10F2

SEQ ID NO: 18: Amino acid sequence of the variable region of the light chain of 2P10F2

SEQ ID NO: 19: Nucleotide sequence of the variable region of the heavy chain of 2P10F2

SEQ ID NO. 20: Amino acid sequence of the variable region of the heavy chain of 2P10F2

SEQ ID NO. 21: Amino acid sequence of CDRL1 of 2P10F2

SEQ ID NO. 22: Amino acid sequence of CDRL2 of 2P10F2

SEQ ID NO. 23: Amino acid sequence of CDRL3 of 2P10F2

SEQ ID NO. 24: Amino acid sequence of CDRH1 of 2P10F2

SEQ ID NO. 25: Amino acid sequence of CDRH2 of 2P10F2

SEQ ID NO. 26: Amino acid sequence of CDRH3 of 2P10F2

SEQ ID NO: 27: DNA fragment containing the DNA sequence encoding the human light chain message sequence and the human kappa chain constant region

SEQ ID NO: 28: DNA fragment containing the DNA sequence encoding the human heavy chain message sequence and the amino acids of the human IgG1 constant region

SEQ ID NO: 29: DNA fragment containing the DNA sequence encoding the human heavy chain message sequence and the amino acids of the human IgG2 constant region

SEQ ID NO: 30: Nucleotide sequence of the light chain of human chimeric LN22R8

SEQ ID NO: 31: Amino acid sequence of light chain of human chimeric LN22R8

SEQ ID NO: 32: Nucleotide sequence of heavy chain IgG1 type of human chimeric LN22R8

SEQ ID NO: 33: Amino acid sequence of heavy chain IgG1 type of human chimeric LN22R8

SEQ ID NO: 34: Nucleotide sequence of heavy chain IgG2 type of human chimeric LN22R8

SEQ ID NO: 35: Amino acid sequence of heavy chain IgG2 type of human chimeric LN22R8

SEQ ID NO: 36: DNA fragment containing the DNA sequence encoding the amino acid sequence of the heavy chain IgG4P type of human chimeric LN22R8

SEQ ID NO: 37: Amino acid sequence of heavy chain IgG4P type of human chimeric LN22R8

SEQ ID NO: 38: DNA fragment containing the DNA sequence encoding the human heavy chain message sequence and the amino acids of the human IgG1 LALA constant region

SEQ ID NO: 39: DNA fragment containing the DNA sequence encoding the human heavy chain message sequence and the amino acids of the human IgG4P constant region

SEQ ID NO: 40: Nucleotide sequence of light chain of human chimeric 2P10F2

SEQ ID NO: 41: Amino acid sequence of light chain of human chimeric 2P10F2

SEQ ID NO: 42: Nucleotide sequence of heavy chain IgG1 LALA type of human chimeric 2P10F2

SEQ ID NO: 43: Amino acid sequence of heavy chain IgG1 LALA type of human chimeric 2P10F2

SEQ ID NO: 44: Nucleotide sequence of heavy chain IgG2 type of human chimeric 2P10F2

SEQ ID NO: 45: Amino acid sequence of heavy chain IgG2 type of human chimeric 2P10F2

SEQ ID NO: 46: Nucleotide sequence of heavy chain IgG4P type of human chimeric 2P10F2

SEQ ID NO: 47: Amino acid sequence of heavy chain IgG4P type of human chimeric 2P10F2

SEQ ID NO: 48: Nucleotide sequence of the variable region of the light chain of rat_CD147_#84 (r#84)

Sequence ID No. 49: Amino acid sequence of the variable region of the light chain of rat_CD147_#84 (r#84)

Sequence ID 50: Nucleotide sequence of the variable region of the heavy chain of rat_CD147_#84 (r#84)

Sequence ID No. 51: Amino acid sequence of the variable region of the heavy chain of rat_CD147_#84 (r#84)

Sequence ID 52: Amino acid sequence of CDRL1 of rat_CD147_#84 (r#84)

Sequence ID 53: Amino acid sequence of CDRL2 of rat_CD147_#84 (r#84)

Sequence ID 54: Amino acid sequence of CDRL3 of rat_CD147_#84 (r#84)

Sequence ID 55: Amino acid sequence of CDRH1 of rat_CD147_#84(r#84)

Sequence ID 56: Amino acid sequence of CDRH2 of rat_CD147_#84(r#84)

Sequence ID 57: Amino acid sequence of CDRH3 of rat_CD147_#84(r#84)

Sequence ID 58: Nucleotide sequence of the variable region of the light chain of rat_CD147_#101 (r#101)

Sequence ID No. 59: Amino acid sequence of the variable region of the light chain of rat_CD147_#101 (r#101)

Sequence ID 60: Nucleotide sequence of the variable region of the heavy chain of rat_CD147_#101 (r#101)

Sequence ID No. 61: Amino acid sequence of the variable region of the heavy chain of rat_CD147_#101 (r#101)

Sequence ID No. 62: Amino acid sequence of CDRL1 of rat_CD147_#101 (r#101)

Sequence ID 63: Amino acid sequence of CDRL2 of rat_CD147_#101 (r#101)

Sequence ID 64: Amino acid sequence of CDRL3 of rat_CD147_#101 (r#101)

Sequence ID 65: Amino acid sequence of CDRH1 of rat_CD147_#101 (r#101)

Sequence ID 66: Amino acid sequence of CDRH2 of rat_CD147_#101 (r#101)

Sequence ID 67: Amino acid sequence of CDRH3 of rat_CD147_#101 (r#101)

SEQ ID NO: 68: Nucleotide sequence of the variable region of the light chain of rat_CD147_#110 (r#110)

Sequence ID No. 69: Amino acid sequence of the variable region of the light chain of rat_CD147_#110 (r#110)

SEQ ID NO: 70: Nucleotide sequence of the variable region of the heavy chain of rat_CD147_#110 (r#110)

Sequence ID 71: Amino acid sequence of the variable region of the heavy chain of rat_CD147_#110 (r#110)

Sequence ID 72: Amino acid sequence of CDRL1 of rat_CD147_#110 (r#110)

Sequence ID 73: Amino acid sequence of CDRL2 of rat_CD147_#110 (r#110)

Sequence ID 74: Amino acid sequence of CDRL3 of rat_CD147_#110 (r#110)

Sequence ID 75: Amino acid sequence of CDRH1 of rat_CD147_#110 (r#110)

Sequence ID 76: Amino acid sequence of CDRH2 of rat_CD147_#110 (r#110)

Sequence ID 77: Amino acid sequence of CDRH3 of rat_CD147_#110 (r#110)

SEQ ID NO: 78: Nucleotide sequence of the variable region of the light chain of rat_CD147_#131 (r#131)

SEQ ID NO: 79: Amino acid sequence of the variable region of the light chain of rat_CD147_#131 (r#131)

Sequence ID No. 80: Nucleotide sequence of the variable region of the heavy chain of rat_CD147_#131 (r#131)

Sequence ID No. 81: Amino acid sequence of the variable region of the heavy chain of rat_CD147_#131 (r#131)

Sequence ID No. 82: Amino acid sequence of CDRL1 of rat_CD147_#131 (r#131)

Sequence ID No. 83: Amino acid sequence of CDRL2 of rat_CD147_#131 (r#131)

Sequence ID No. 84: Amino acid sequence of CDRL3 of rat_CD147_#131 (r#131)

Sequence ID No. 85: Amino acid sequence of CDRH1 of rat_CD147_#131 (r#131)

Sequence ID No. 86: Amino acid sequence of CDRH2 of rat_CD147_#131 (r#131)

Sequence ID No. 87: Amino acid sequence of CDRH3 of rat_CD147_#131 (r#131)

SEQ ID NO: 88: DNA fragment containing the DNA sequence encoding the human heavy chain message sequence and the amino acids of the human IgG4PFALA constant region

SEQ ID NO: 89: Nucleotide sequence of the light chain of human chimeric rat_CD147_#84

SEQ ID NO: 90: Amino acid sequence of light chain of human chimeric rat_CD147_#84

SEQ ID NO: 91: Nucleotide sequence of heavy chain IgG1 type of human chimeric rat_CD147_#84

SEQ ID NO: 92: Amino acid sequence of heavy chain IgG1 type of human chimeric rat_CD147_#84

SEQ ID NO: 93: Nucleotide sequence of heavy chain IgG2 type of human chimeric rat_CD147_#84

SEQ ID NO: 94: Amino acid sequence of heavy chain IgG2 type of human chimeric rat_CD147_#84

SEQ ID NO: 95: Nucleotide sequence of heavy chain IgG4P type of human chimeric rat_CD147_#84

SEQ ID NO: 96: Amino acid sequence of heavy chain IgG4P type of human chimeric rat_CD147_#84

SEQ ID NO: 97: Nucleotide sequence of heavy chain IgG1 LALA type of human chimeric rat_CD147_#84

SEQ ID NO: 98: Amino acid sequence of heavy chain IgG1 LALA type of human chimeric rat_CD147_#84

SEQ ID NO: 99: Nucleotide sequence of heavy chain IgG4PFALA type of human chimeric rat_CD147_#84

SEQ ID NO: 100: Amino acid sequence of heavy chain IgG4PFALA type of human chimeric rat_CD147_#84

SEQ ID NO: 101: Nucleotide sequence of the light chain of human chimeric rat_CD147_#101

SEQ ID NO: 102: Amino acid sequence of the light chain of human chimeric rat_CD147_#101

SEQ ID NO: 103: Nucleotide sequence of heavy chain IgG2 of human chimeric rat_CD147_#101

SEQ ID NO: 104: Amino acid sequence of heavy chain IgG2 of human chimeric rat_CD147_#101

SEQ ID NO: 105: Nucleotide sequence of heavy chain IgG4P of human chimeric rat_CD147_#101

SEQ ID NO: 106: Amino acid sequence of heavy chain IgG4P of human chimeric rat_CD147_#101

SEQ ID NO: 107: Nucleotide sequence of heavy chain IgG4PFALA of human chimeric rat_CD147_#101

SEQ ID NO: 108: Amino acid sequence of heavy chain IgG4PFALA of human chimeric rat_CD147_#101

SEQ ID NO: 109: Nucleotide sequence of the light chain of human chimeric rat_CD147_#110

SEQ ID NO: 110: Amino acid sequence of the light chain of human chimeric rat_CD147_#110

SEQ ID NO: 111: Nucleotide sequence of heavy chain IgG2 of human chimeric rat_CD147_#110

SEQ ID NO: 112: Amino acid sequence of heavy chain IgG2 of human chimeric rat_CD147_#110

SEQ ID NO: 113: Nucleotide sequence of heavy chain IgG4P of human chimeric rat_CD147_#110

SEQ ID NO: 114: Amino acid sequence of heavy chain IgG4P of human chimeric rat_CD147_#110

SEQ ID NO: 115: Nucleotide sequence of heavy chain IgG4PFALA of human chimeric rat_CD147_#110

SEQ ID NO: 116: Amino acid sequence of heavy chain IgG4PFALA of human chimeric rat_CD147_#110

SEQ ID NO: 117: Nucleotide sequence of the light chain of human chimeric rat_CD147_#131

SEQ ID NO: 118: Amino acid sequence of light chain of human chimeric rat_CD147_#131

SEQ ID NO: 119: Nucleotide sequence of heavy chain IgG2 of human chimeric rat_CD147_#131

SEQ ID NO: 120: Amino acid sequence of heavy chain IgG2 of human chimeric rat_CD147_#131

SEQ ID NO: 121: Nucleotide sequence of heavy chain IgG4P of human chimeric rat_CD147_#131

SEQ ID NO: 122: Amino acid sequence of heavy chain IgG4P of human chimeric rat_CD147_#131

SEQ ID NO: 123: Amino acid sequence of #84H1hIgG2

SEQ ID NO: 124: Nucleotide sequence of #84H1hIgG2

SEQ ID NO: 125: Amino acid sequence of #84H1hIgG4P

SEQ ID NO: 126: Nucleotide sequence of #84H1hIgG4P

SEQ ID NO: 127: Amino acid sequence of #84L2h

SEQ ID NO: 128: Nucleotide sequence of #84L2h

SEQ ID NO: 129: Amino acid sequence of #101H1hIgG2

SEQ ID NO: 130: Nucleotide sequence of #101H1hIgG2

Sequence ID 131: Amino acid sequence of #101H1hIgG4P

SEQ ID NO: 132: Nucleotide sequence of #101H1hIgG4P

SEQ ID NO: 133: Amino acid sequence of #101L2h

SEQ ID NO: 134: Nucleotide sequence of #101L2h

SEQ ID NO: 135: Amino acid sequence of #110H1hIgG4P

SEQ ID NO: 136: Nucleotide sequence of #110H1hIgG4P

SEQ ID NO: 137: Amino acid sequence of #110L4h

SEQ ID NO: 138: Nucleotide sequence of #110L4h

SEQ ID NO: 139: Amino acid sequence of #131H2hIgG2

SEQ ID NO: 140: Nucleotide sequence of #131H2hIgG2

SEQ ID NO: 141: Amino acid sequence of #131L2h

SEQ ID NO: 142: Nucleotide sequence of #131L2h

SEQ ID NO: 143: mu3 region of human CD147v1

Sequence ID 144: mu3 region of cynomolgus CD147

SEQ ID NO: 145: Amino acid sequence of human KLF5

SEQ ID NO: 146: Nucleotide sequence of human KLF5

Sequence ID 147: Amino acid sequence of #110H13hIgG4P

SEQ ID NO: 148: Nucleotide sequence of #110H13hIgG4P

SEQ ID NO: 149: Amino acid sequence of #110L2h

SEQ ID NO: 150: Nucleotide sequence of #110L2h

Sequence ID 151: Amino acid sequence of #110L12h

SEQ ID NO: 152: Nucleotide sequence of #110L12h

<110> DAIICHI SANKYO COMPANY,LIMITED

<120> Anti-CD147 antibodies, uses and manufacturing methods thereof

<130> DSPCT-FP1821

<140>TW 107125910

<141> 2018-07-26

<150> JP2017-145701

<151> 2017-07-27

<160> 152

<170> PatentIn version 3.5

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<223> Human Chimeric 2P10F2_Heavy Chain_IgG4P

<400> 47

<210> 48

<211> 324

<212> DNA

<213> Brown Rat

<400> 48

<210> 49

<211> 108

<212> PRT

<213> Brown Rat

<400> 49

<210> 50

<211> 363

<212> DNA

<213> Brown Rat

<400> 50

<210> 51

<211> 121

<212> PRT

<213> Brown Rat

<400> 51

<210> 52

<211> 11

<212> PRT

<213> Brown Rat

<400> 52

<210> 53

<211> 7

<212> PRT

<213> Brown Rat

<400> 53

<210> 54

<211> 9

<212> PRT

<213> Brown Rat

<400> 54

<210> 55

<211> 10

<212> PRT

<213> Brown Rat

<400> 55

<210> 56

<211> 10

<212> PRT

<213> Brown Rat

<400> 56

<210> 57

<211> 12

<212> PRT

<213> Brown Rat

<400> 57

<210> 58

<211> 324

<212> DNA

<213> Brown Rat

<400> 58

<210> 59

<211> 108

<212> PRT

<213> Brown Rat

<400> 59

<210> 60

<211> 354

<212> DNA

<213> Brown Rat

<400> 60

<210> 61

<211> 118

<212> PRT

<213> Brown Rat

<400> 61

<210> 62

<211> 11

<212> PRT

<213> Brown Rat

<400> 62

<210> 63

<211> 7

<212> PRT

<213> Brown Rat

<400> 63

<210> 64

<211> 9

<212> PRT

<213> Brown Rat

<400> 64

<210> 65

<211> 10

<212> PRT

<213> Brown Rat

<400> 65

<210> 66

<211> 10

<212> PRT

<213> Brown Rat

<400> 66

<210> 67

<211> 9

<212> PRT

<213> Brown Rat

<400> 67

<210> 68

<211> 324

<212> DNA

<213> Brown Rat

<400> 68

<210> 69

<211> 108

<212> PRT

<213> Brown Rat

<400> 69

<210> 70

<211> 351

<212> DNA

<213> Brown Rat

<400> 70

<210> 71

<211> 117

<212> PRT

<213> Brown Rat

<400> 71

<210> 72

<211> 11

<212> PRT

<213> Brown Rat

<400> 72

<210> 73

<211> 7

<212> PRT

<213> Brown Rat

<400> 73

<210> 74

<211> 9

<212> PRT

<213> Brown Rat

<400> 74

<210> 75

<211> 10

<212> PRT

<213> Brown Rat

<400> 75

<210> 76

<211> 10

<212> PRT

<213> Brown Rat

<400> 76

<210> 77

<211> 8

<212> PRT

<213> Brown Rat

<400> 77

<210> 78

<211> 324

<212> DNA

<213> Brown Rat

<400> 78

<210> 79

<211> 108

<212> PRT

<213> Brown Rat

<400> 79

<210> 80

<211> 357

<212> DNA

<213> Brown Rat

<400> 80

<210> 81

<211> 119

<212> PRT

<213> Brown Rat

<400> 81

<210> 82

<211> 11

<212> PRT

<213> Brown Rat

<400> 82

<210> 83

<211> 7

<212> PRT

<213> Brown Rat

<400> 83

<210> 84

<211> 9

<212> PRT

<213> Brown Rat

<400> 84

<210> 85

<211> 10

<212> PRT

<213> Brown Rat

<400> 85

<210> 86

<211> 10

<212> PRT

<213> Brown Rat

<400> 86

<210> 87

<211> 10

<212> PRT

<213> Brown Rat

<400> 87

<210> 88

<211> 1117

<212> DNA

<213> Artificial sequence

<220>

<223> DNA containing DNA sequences encoding human heavy chain message sequences and amino acids of the human IgG4PFALA consensus region

<400> 88

<210> 89

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Light Chain

<400> 89

<210> 90

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Light Chain

<400> 90

<210> 91

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG1

<400> 91

<210> 92

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG1

<400> 92

<210> 93

<211> 1398

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG2

<400> 93

<210> 94

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG2

<400> 94

<210> 95

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG4P

<400> 95

<210> 96

<211> 467

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG4P

<400> 96

<210> 97

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG1LALA

<400> 97

<210> 98

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG1LALA

<400> 98

<210> 99

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG4PFALA

<400> 99

<210> 100

<211> 467

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 84_Heavy Chain_IgG4PFALA

<400> 100

<210> 101

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> Human chimeric rat 101_light chain

<400> 101

<210> 102

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> Human chimeric rat 101_light chain

<400> 102

<210> 103

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 101_Heavy Chain_IgG2

<400> 103

<210> 104

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 101_Heavy Chain_IgG2

<400> 104

<210> 105

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 101_Heavy Chain_IgG4P

<400> 105

<210> 106

<211> 464

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 101_Heavy Chain_IgG4P

<400> 106

<210> 107

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 101_Heavy Chain_IgG4PFALA

<400> 107

<210> 108

<211> 464

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 101_Heavy Chain_IgG4PFALA

<400> 108

<210> 109

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Light Chain

<400> 109

<210> 110

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Light Chain

<400> 110

<210> 111

<211> 1386

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Heavy Chain_IgG2

<400> 111

<210> 112

<211> 462

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Heavy Chain_IgG2

<400> 112

<210> 113

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Heavy Chain_IgG4P

<400> 113

<210> 114

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Heavy Chain_IgG4P

<400> 114

<210> 115

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Heavy Chain_IgG4PFALA

<400> 115

<210> 116

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 110_Heavy Chain_IgG4PFALA

<400> 116

<210> 117

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> Human chimeric rat 131_light chain

<400> 117

<210> 118

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> Human chimeric rat 131_light chain

<400> 118

<210> 119

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 131_Heavy Chain_IgG2

<400> 119

<210> 120

<211> 464

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 131_Heavy Chain_IgG2

<400> 120

<210> 121

<211> 1395

<212> DNA

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 131_Heavy Chain_IgG4P

<400> 121

<210> 122

<211> 465

<212> PRT

<213> Artificial sequence

<220>

<223> Human Chimeric Rat 131_Heavy Chain_IgG4P

<400> 122

<210> 123

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> #84H1hIgG2

<400> 123

<210> 124

<211> 1398

<212> DNA

<213> Artificial sequence

<220>

<223> #84H1hIgG2

<400> 124

<210> 125

<211> 467

<212> PRT

<213> Artificial sequence

<220>

<223> #84H1hIgG4P

<400> 125

<210> 126

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> #84H1hIgG4P

<400> 126

<210> 127

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> #84L2h

<400> 127

<210> 128

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> #84L2h

<400> 128

<210> 129

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> #101H1hIgG2

<400> 129

<210> 130

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> #101H1hIgG2

<400> 130

<210> 131

<211> 464

<212> PRT

<213> Artificial sequence

<220>

<223> #101H1hIgG4P

<400> 131

<210> 132

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> #101H1hIgG4P

<400> 132

<210> 133

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> #101L2h

<400> 133

<210> 134

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> #101L2h

<400> 134

<210> 135

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> #110H1hIgG4P

<400> 135

<210> 136

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> #110H1hIgG4P

<400> 136

<210> 137

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> #110L4h

<400> 137

<210> 138

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> #110L4h

<400> 138

<210> 139

<211> 464

<212> PRT

<213> Artificial sequence

<220>

<223> #131H2hIgG2

<400> 139

<210> 140

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> #131H2hIgG2

<400> 140

<210> 141

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> #131L2h

<400> 141

<210> 142

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> #131L2h

<400> 142

<210> 143

<211> 17

<212> PRT

<213> Homo sapiens

<400> 143

<210> 144

<211> 17

<212> PRT

<213> Crab-eating macaque

<400> 144

<210> 145

<211> 457

<212> PRT

<213> Homo sapiens

<400> 145

<210> 146

<211> 1374

<212> DNA

<213> Homo sapiens

<400> 146

<210> 147

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> #110H13hIgG4P

<400> 147

<210> 148

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> #110H13hIgG4P

<400> 148

<210> 149

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> #110L2h

<400> 149

<210> 150

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> #110L2h

<400> 150

<210> 151

<211> 234

<212> PRT

<213> Artificial sequence

<220>

<223> #110L12h

<400> 151

<210> 152

<211> 702

<212> DNA

<213> Artificial sequence

<220>

<223> #110L12h

<400> 152

Claims (70)

An anti-human CD147 antibody or an antigen-binding fragment thereof, which includes a heavy chain sequence and a light chain sequence, wherein: (A) in the heavy chain sequence, it includes a variable region with CDRH1, CDRH2 and CDRH3, the aforementioned CDRH1 is composed of the sequence The aforementioned CDRH2 is composed of the amino acid sequence represented by the sequence identification number 75, and the aforementioned CDRH3 is composed of the amino acid sequence represented by the sequence identification number 77; and The light chain sequence includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 72, and the aforementioned CDRL2 is composed of the amino group represented by SEQ ID NO: 73 The aforementioned CDRL3 is composed of the amino acid sequence shown in Sequence ID No. 74; (B) In the heavy chain sequence, it includes a variable region having CDRH1, CDRH2 and CDRH3, and the aforementioned CDRH1 is composed of the sequence The aforementioned CDRH2 is composed of the amino acid sequence represented by the sequence identification number 55, and the aforementioned CDRH3 is composed of the amino acid sequence represented by the sequence identification number 57; and The light chain sequence includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 52, and the aforementioned CDRL2 is composed of the amino group represented by SEQ ID NO: 53 Composed of an acid sequence, the aforementioned CDRL3 is composed of the amino acid sequence shown in Sequence ID No. 54; (C) in the heavy chain sequence, it includes a variable region having CDRH1, CDRH2 and CDRH3, the aforementioned CDRH1 is composed of the sequence Identification number The aforementioned CDRH2 is composed of the amino acid sequence represented by Sequence ID No. 66, and the aforementioned CDRH3 is composed of the amino acid sequence represented by Sequence ID No. 67; and in light In the chain sequence, it includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 62, and the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 63 Composed of, the aforementioned CDRL3 is composed of the amino acid sequence shown in SEQ ID NO: 64; or (D) in the heavy chain sequence, it includes a variable region having CDRH1, CDRH2 and CDRH3, the aforementioned CDRH1 is identified by the sequence The aforementioned CDRH2 is composed of the amino acid sequence represented by Sequence No. 85, the aforementioned CDRH3 is composed of the amino acid sequence represented by Sequence No. 87; and The light chain sequence includes a variable region having CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 82, and the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 83 The aforementioned CDRL3 is composed of the amino acid sequence shown in Sequence ID No. 84. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has reduced or missing ADCC activity. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has reduced or missing CDC activity. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has reduced or missing ADCP activity. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1, It binds to an epitope comprising residues from arginine (Arg) No. 106 to glycine (Gly) No. 165 of SEQ ID NO. 3. For example, the anti-human CD147 antibody or antigen-binding fragment thereof in claim 1, which contains arginine (Arg) No. 106 and lysine (Lys) No. 108 in the amino acid sequence recorded in Sequence ID No. 3 ), No. 109 alanine (Ala), No. 110 valine (Val), No. 127 lysine (Lys), No. 128 serine (Ser), No. 129 gluten Amino acid (Glu), No. 130 Serine (Ser), No. 131 Valine (Val), No. 132 Proline (Pro), No. 133 Proline (Pro), Antitope binding of each residue of valine (Val) No. 134, glutamic acid (Gln) No. 164, and glycine (Gly) No. 165. The anti-human CD147 antibody or antigen-binding fragment thereof of Claim 1, wherein the heavy chain sequence includes a variable region having CDRH1, CDRH2 and CDRH3, and the aforementioned CDRH1 is the amino acid sequence represented by SEQ ID NO: 75 Composed of, the aforementioned CDRH2 is composed of the amino acid sequence represented by SEQ ID NO: 76, the aforementioned CDRH3 is composed of the amino acid sequence represented by SEQ ID NO: 77; and in the light chain sequence, it includes CDRL1 , the variable regions of CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 72, the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 73, and the aforementioned CDRL3 is composed of the sequence It consists of the amino acid sequence shown by identification number 74. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1, which contains the amino acid sequence recorded in Sequence ID No. 143, or is in the sequence The sequence of column ID 143 has one or several amino acids deleted, substituted or added, and is bound to an epitope of an amino acid sequence. The anti-human CD147 antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain sequence includes a variable region having CDRH1, CDRH2 and CDRH3, and the aforementioned CDRH1 is an amine group represented by SEQ ID NO: 55 Composed of an acid sequence, the aforementioned CDRH2 is composed of an amino acid sequence represented by SEQ ID NO: 56, and the aforementioned CDRH3 is composed of an amino acid sequence represented by SEQ ID NO: 57; and in the light chain sequence, it includes It has variable regions of CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 52, the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 53, and the aforementioned CDRL3 is composed of the amino acid sequence represented by SEQ ID NO: 53. It consists of the amino acid sequence represented by SEQ ID NO: 54. The anti-human CD147 antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain sequence includes a variable region having CDRH1, CDRH2 and CDRH3, and the aforementioned CDRH1 is the amino acid represented by SEQ ID NO: 65 The aforementioned CDRH2 is composed of the amino acid sequence represented by SEQ ID NO: 66, and the aforementioned CDRH3 is composed of the amino acid sequence represented by SEQ ID NO: 67; and in the light chain sequence, it includes The variable regions of CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 62, the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 63, and the aforementioned CDRL3 is composed of sequence It consists of the amino acid sequence shown in column ID 64. The anti-human CD147 antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain sequence includes a variable region having CDRH1, CDRH2, and CDRH3, and the aforementioned CDRH1 is the amino acid represented by SEQ ID NO: 85 The aforementioned CDRH2 is composed of the amino acid sequence represented by SEQ ID NO: 86, and the aforementioned CDRH3 is composed of the amino acid sequence represented by SEQ ID NO: 87; and in the light chain sequence, it contains The variable regions of CDRL1, CDRL2 and CDRL3, the aforementioned CDRL1 is composed of the amino acid sequence represented by SEQ ID NO: 82, the aforementioned CDRL2 is composed of the amino acid sequence represented by SEQ ID NO: 83, and the aforementioned CDRL3 is composed of It consists of the amino acid sequence shown in SEQ ID NO: 84. The anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 is selected from the group consisting of Fab, F(ab')2, Fab' and Fv. The anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 is a scFv. The anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 is a chimeric antibody. The anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 is humanized. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the heavy chain of which includes a human immunoglobulin G1 heavy chain, a human immunoglobulin G2 heavy chain or a human immunoglobulin G4 heavy chain. The constant region, light chain includes the constant region of the human immunoglobulin kappa light chain. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 16, the heavy chain thereof includes the constant region of the human immunoglobulin G4 heavy chain. The anti-human CD147 antibody or antigen-binding fragment thereof of claim 17, wherein in the constant region of the human immunoglobulin G4 heavy chain, the serine (Ser) No. 228 shown in the EU index (EU index) is passed through proline Acid (Pro) substitution. For example, in the anti-human CD147 antibody or antigen-binding fragment thereof of claim 17, in the constant region of the human immunoglobulin G4 heavy chain, the phenylalanine (Phe) in No. 234 shown in the EU index is replaced by alanine (Ala). ), No. 235’s leucine (Leu) was replaced by alanine (Ala). For example, in the anti-human CD147 antibody or antigen-binding fragment thereof of claim 17, in the constant region of the human immunoglobulin G4 heavy chain, serine (Ser) No. 228 shown in the EU index is replaced by proline. (Pro), the phenylalanine (Phe) in No. 234 was replaced by alanine (Ala), and the leucine (Leu) in No. 235 was replaced by alanine (Ala). The anti-human CD147 antibody or antigen-binding fragment thereof of claim 16, wherein the heavy chain comprises the constant region of the human immunoglobulin G2 heavy chain. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has the following (c) and (d), and will be activated through CD147 signaling, and (c) is selected from the group consisting of the following (c1)~( The heavy chain variable region described in any one of the groups c2): (c1) The amino acid sequence represented by SEQ ID NO: 135 A heavy chain variable region composed of amino acid residues No. 20 to 136; and (c2) a heavy chain variable region composed of amino acid residues Nos. 20 to 136 of the amino acid sequence shown in Sequence ID No. 147 The heavy chain variable region, and (d) the light chain variable region described in any one selected from the group including the following (d1) to (d3): (d1) the amino group represented by SEQ ID NO: 137 The light chain variable region composed of amino acid residues No. 21 to 128 of the acid sequence; (d2) Amino acid residues No. 21 to 128 of the amino acid sequence shown in Sequence ID No. 149 The light chain variable region composed of; and (d3) the light chain variable region composed of amino acid residues No. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 151. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of Claim 22, which includes a heavy chain variable region composed of amino acid residues No. 20 to No. 136 of the amino acid sequence shown in Sequence ID No. 135; And a light chain variable region composed of amino acid residues No. 21 to No. 128 of the amino acid sequence shown in SEQ ID NO: 137. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to claim 22, which includes a heavy chain composed of amino acid residues No. 20 to No. 463 of the amino acid sequence shown in Sequence ID No. 135; and consists of the sequence A light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence represented by identification number 137. Such as the anti-human CD147 antibody or antigen-binding fragment thereof of claim 22, which includes The heavy chain variable region composed of amino acid residues Nos. 20 to 136 of the amino acid sequence represented by SEQ ID NO: 147; and the heavy chain variable region composed of amino acid residues Nos. 21 to 136 of the amino acid sequence represented by SEQ ID NO: 149 The light chain variable region composed of amino acid residue No. 128. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 22, which includes a heavy chain composed of amino acid residues No. 20 to No. 463 of the amino acid sequence shown in Sequence ID No. 147; and consists of the sequence A light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence represented by identification number 149. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 22, which includes a heavy chain variable region composed of amino acid residues No. 20 to No. 136 of the amino acid sequence shown in Sequence ID No. 147; And a light chain variable region composed of amino acid residues No. 21 to No. 128 of the amino acid sequence shown in SEQ ID NO: 151. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 22, which includes a heavy chain composed of amino acid residues No. 20 to No. 463 of the amino acid sequence shown in Sequence ID No. 147; and consists of the sequence A light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence represented by identification number 151. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has the following (a) and (b) and will be activated through CD147 signaling, (a) The heavy chain variable region described in any one of the following groups (a1) to (a4): (a1) Nos. 20 to 140 of the amino acid sequence represented by Sequence ID No. 123 A heavy chain variable region composed of amino acid residues No. 125; and (a2) a heavy chain composed of amino acid residues Nos. 20 to 140 of the amino acid sequence shown in Sequence ID No. 125 may variable region, and (b) a light chain variable region composed of amino acid residues Nos. 21 to 128 of the amino acid sequence shown in SEQ ID NO: 127. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of Claim 29, which includes: a heavy chain variable region composed of amino acid residues No. 20 to No. 140 of the amino acid sequence shown in Sequence ID No. 123 Or the heavy chain variable region composed of amino acid residues No. 20 to 140 of the amino acid sequence shown in SEQ ID NO: 125; and No. 21 of the amino acid sequence shown in SEQ ID NO: 127 The light chain variable region composed of amino acid residues ~128. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 29, which includes: a heavy chain composed of amino acid residues No. 20 to No. 466 of the amino acid sequence shown in Sequence ID No. 123 or a heavy chain composed of the sequence A heavy chain composed of amino acid residues No. 20 to 467 of the amino acid sequence shown in SEQ ID No. 125; and a heavy chain composed of amino acid residues No. 21 to 234 of the amino acid sequence shown in SEQ ID NO. 127 A light chain composed of acid residues. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has the following (e) and (f), and will be activated through CD147 message transduction, and (e) is selected from the group consisting of the following (e1)~( The heavy chain variable region described in any one of the groups e2): (e1) The heavy chain composed of amino acid residues No. 20 to 137 of the amino acid sequence represented by Sequence ID No. 129 may variable region; and (e2) a heavy chain variable region consisting of amino acid residues No. 20 to 137 of the amino acid sequence represented by SEQ ID NO: 131, and (f) a heavy chain variable region represented by SEQ ID NO: 133 The light chain variable region is composed of amino acid residues No. 21 to 128 of the amino acid sequence shown. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of Claim 32, which includes: a heavy chain variable region composed of amino acid residues No. 20 to No. 137 of the amino acid sequence shown in Sequence ID No. 129 Or the heavy chain variable region composed of amino acid residues Nos. 20 to 137 of the amino acid sequence shown in SEQ ID NO: 131; and No. 21 of the amino acid sequence shown in SEQ ID NO: 133 The light chain variable region composed of amino acid residues ~128. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of Claim 32, which includes: a heavy chain composed of amino acid residues No. 20 to No. 463 of the amino acid sequence shown in Sequence ID No. 129 or a heavy chain composed of the sequence Composed of amino acid residues No. 20 to No. 464 of the amino acid sequence represented by identification number 131 A heavy chain; and a light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in Sequence ID No. 133. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 1 has the following (g) and (h), and will be activated through CD147 signaling, (g) the amine group represented by Sequence ID No. 139 The heavy chain variable region composed of amino acid residues No. 20 to 138 of the acid sequence, and (h) the amino acid residues No. 21 to 128 of the amino acid sequence represented by SEQ ID NO. 141 light chain variable region. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to claim 35, which includes: a heavy chain variable region composed of amino acid residues No. 20 to No. 138 of the amino acid sequence shown in SEQ ID NO: 139 ; And a light chain variable region composed of amino acid residues No. 21 to No. 128 of the amino acid sequence shown in Sequence ID No. 141. For example, the anti-human CD147 antibody or antigen-binding fragment thereof in claim 35, which includes: a heavy chain composed of amino acid residues No. 20 to 464 of the amino acid sequence shown in Sequence ID No. 139; and A light chain composed of amino acid residues No. 21 to No. 234 of the amino acid sequence shown in Sequence ID No. 141. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 22 to 37 has a reduced or deleted ADCC activity. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 22 to 37 has a reduced or deleted CDC activity. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 22 to 37 has a reduced or deleted ADCP activity. A pharmaceutical composition containing at least any one of the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40. Such as the pharmaceutical composition of claim 41, which is an anti-tumor agent. The pharmaceutical composition of claim 42, wherein the tumor is a tumor expressing CD147. For example, the pharmaceutical composition of claim 42 or 43, wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, lymphoma, thyroid cancer, skin cancer, head and neck cancer , sarcoma, prostate cancer, bladder cancer, brain tumors, gastrointestinal stromal tumors (GIST), leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoid Leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin lymphoma, or diffuse large cell B-cell lymphoma (DLBCL). For example, the pharmaceutical composition of claim 42 or 43, wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, renal cancer, leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell Type B cell lymphoma (DLBCL). For example, the pharmaceutical composition of claim 42 or 43, wherein the tumor is a SMAD4-positive tumor or a tumor with reduced expression of KLF5 or loss of expression of KLF5. For example, the pharmaceutical composition of claim 42 or 43 further contains other anti-tumor agents. A use of the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40 or the pharmaceutical composition according to any one of claims 41 to 47, which is used to produce a therapeutic agent for tumors. The use of claim 48, wherein the tumor is a tumor expressing CD147. Such as the use of claim 48 or 49, wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, lymphoma, thyroid cancer, skin cancer, head and neck cancer, sarcoma , prostate cancer, bladder cancer, brain tumors, gastrointestinal stromal tumors (GIST), leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoid leukemia ( ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell B-cell lymphoma (DLBCL). For example, the use of claim 48 or 49, wherein the tumor is pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, renal cancer, leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL) ), acute lymphoblastic leukemia (ALL), malignant lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma, or diffuse large cell B-cell lymphoma (DLBCL). The use of claim 48 or 49, wherein the tumor is a SMAD4-positive tumor or a tumor in which KLF5 is reduced or deleted. The use of claim 48 or 49, wherein the agent for treating tumors is administered in combination with another anti-tumor agent. A polynucleotide encoding the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40. Such as the polynucleotide of claim 54, which includes: encoding CDRH1 composed of the amino acid sequence recorded in SEQ ID NO: 75, CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 76, and CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 77 Polynucleotides encoding CDRH3 composed of the amino acid sequence recorded in SEQ ID NO: 72; and CDRL1 composed of the amino acid sequence recorded in SEQ ID NO: 72, CDRL2 composed of the amino acid sequence recorded in SEQ ID NO: 73, and CDRL2 composed of the amino acid sequence recorded in SEQ ID NO: 73 The polynucleotide of CDRL3 composed of the amino acid sequence described in SEQ ID NO: 74. Such as the polynucleotide of claim 54, which includes: encoding CDRH1 composed of the amino acid sequence recorded in SEQ ID NO: 55, CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 56, and CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 57 Polynucleotides encoding CDRH3 composed of the amino acid sequence recorded in SEQ ID NO: 52; and CDRL1 composed of the amino acid sequence recorded in SEQ ID NO: 52, CDRL2 composed of the amino acid sequence recorded in SEQ ID NO: 53, and CDRL2 composed of the amino acid sequence recorded in SEQ ID NO: 53 The polynucleotide of CDRL3 composed of the amino acid sequence described in SEQ ID NO: 54. Such as the polynucleotide of claim 54, which includes: Multinucleus encoding CDRH1 composed of the amino acid sequence described in SEQ ID NO: 65, CDRH2 composed of the amino acid sequence described in SEQ ID NO: 66, and CDRH3 composed of the amino acid sequence described in SEQ ID NO: 67 and encoding CDRL1 composed of the amino acid sequence described in SEQ ID NO: 62, CDRL2 composed of the amino acid sequence described in SEQ ID NO: 63, and CDRL2 composed of the amino acid sequence described in SEQ ID NO: 64 CDRL3 polynucleotide. For example, the polynucleotide of claim 54, which includes: encoding CDRH1 composed of the amino acid sequence recorded in SEQ ID NO: 85, CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 86, and CDRH2 composed of the amino acid sequence recorded in SEQ ID NO: 87 Polynucleotides encoding CDRH3 composed of the amino acid sequence recorded in SEQ ID NO: 82; and CDRL1 composed of the amino acid sequence recorded in SEQ ID NO: 82, CDRL2 composed of the amino acid sequence recorded in SEQ ID NO: 83, and CDRL2 composed of the amino acid sequence recorded in SEQ ID NO: 83 The polynucleotide of CDRL3 composed of the amino acid sequence described in SEQ ID NO: 84. An expression vector containing the polynucleotide of any one of claims 54 to 58. A host cell transformed by an expression vector as claimed in claim 59. A method for producing the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, which includes culturing the host cell according to claim 60, and extracting the intended anti-human CD147 antibody or antigen-binding fragment thereof from the culture product Fragment steps. For example, the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1, 4, 22 to 37, wherein the activation of signaling through CD147 is activation of p38MAPK and/or activation of SMAD4. For example, the anti-human CD147 antibody or antigen-binding fragment thereof of claim 62, wherein the activation of p38MAPK and/or the activation of SMAD4 is an increase in the expression of p38MAPK, the phosphorylation of p38MAPK, the phosphorylation of HSP27, and the increase in the expression of CXCL8 , increase in rhoB expression, decrease in KLF5 mRNA or decrease in KLF5 protein expression. A use of the anti-human CD147 antibody or antigen-binding fragment thereof as claimed in claim 62 or 63, which is used to produce a therapeutic agent for tumors. A method of predicting response to cancer treatment, comprising detecting the expression of SMAD4 or the expression of KLF5 in a biological sample derived from a cancer patient; and detecting the expression of SMAD4 or the expression of reduced or absent KLF5 in patients The patient is determined to be responsive to cancer treatment using the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, or the pharmaceutical composition according to any one of claims 41 to 47. A method of screening cancer treatment subjects, which includes detecting the expression of SMAD4 or the expression of KLF5 in a biological sample derived from a cancer patient; and detecting the expression of SMAD4 or the expression of KLF5 in patients who are detected to be reduced or the expression of KLF5 is missing. Patients screened for use as an anti-human CD147 antibody or an antigen thereof according to any one of claims 1 to 40 The object of cancer treatment is a binding fragment or a pharmaceutical composition according to any one of claims 41 to 47. A use of the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40 or the pharmaceutical composition according to any one of claims 41 to 47, which is used to manufacture a medicament for cancer treatment, the Therapeutic agents are used for patients in whom SMAD4 expression is detected using a biological sample derived from a cancer patient, and the presence or absence of expression of SMAD4 contained in the biological sample is detected, or the expression of KLF5 is detected. Patients with reduced expression of KLF5 or lack of expression of KLF5. A kit for determining the effectiveness of cancer treatment using an anti-human CD147 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 40 or a pharmaceutical composition according to any one of claims 41 to 47 A reactive kit comprising at least a means for detecting the expression of SMAD4 or the expression of KLF5 in a biological sample derived from a cancer patient. An antibody-drug complex comprising the anti-human CD147 antibody or antigen-binding fragment thereof according to any one of claims 1 to 40 combined with other drugs. A bispecific antibody (bispecific antibody), which includes an antigen-binding fragment of the anti-human CD147 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 40, and an antigen-binding fragment that binds to an antigen other than CD147.

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